ABSTRACTS. 293

TABLE II.

Interval between I

Month if Year Case. Speci'es. Infection when Symptoms Remarks.

and Symptoms. appeared.

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

I. Dog At least 24 days October -2.

" 34 days " -

3· " 79-85 days November -4· " 65 days "

Bitten by same dog as 1 and 2.

5· " 7~ months (in March (1919) -quarantine)

6. Bovine 22 days December

tAll 7· " 23 ,. " bitten by 8. " 27 " "

f same dog.

9· " 27 " " 10. " 34 " " II. " 84 "

February (1919) -

The distances covered, as the crow flies, during their run in the case of eight dogs, about which reasonably accurate information was available, was .as follows: In one case 20 miles, but the dog was shot while still going strong because it bit a man; in another case 8 miles, but was then shot because it had bitten two men and seven dogs; in another case 20 miles, when it was shot; in another case 12 miles, when it was captured hy a policeman whom it bit-it died in captivity; in another case 22 miles. when ~t was captured and shot for attacking ducks; in another case 30 miles, when it was shot while still going strong; in another case 12 miles, when, after biting ten people, it returned to its home and died two days afterwards; in another case 30 miles, and died exhausted. (From the Annual Report of the Chief. Veterinary Officer of the Board if Agriculture and Fisheries for the year 1918.)

OBSERVATIONS AND EXPERIM,.ENTS WITH REGARD TO CONTAGIOUS ABORTION IN CATTLE.

Occurrence of the Disease in South Africa.-The existence of contagious .abortion amongst Gattle in South Africa was definitely proved for the first time in 1913. Until then, though the presence of the disease was generally admitted and it had caused considerable losses to stock-owners during many years, it had not been settled beyond doubt that the disease was due to the Bacillus abortus of Bang. References to abortion in cows, presumed in the absence of proof to be contagious abortion, have been made from time to time in the Agricultura,l Journals of the Cape of Good Hope and

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Transvaal, and in the Annual Reports of the Chief Veterinary Surgeons for these two provinces. A popular bulletin by Mr C. E. Gray, Chief Veterinary Surgeon of the Transvaal, was written for distribution amongst farmers (Agricultural Bulletin, No.6, 1906). In this he refers to a severe outbreak of contagious abortion in the vicinity of Johannesburg, which wa,s traced to the introduction of an infected cow into the herd. The article was mainly devoted to preventive measures and treatment, and a full description of which was given. The number of outbreaks which occurred during the year cnding March 1916 was twenty-six, and for the year ending March 1917, forty-two. Outbreaks have only been recorded since the introduction of the agglutination test in 1914 as a means of diagnosing the disease. It is only of late, since suspected herds in the Union have been subjected to the serological tests, that it has been seen how very widespread the disease is. Positive results have been obtained from most parts of the Transvaal, Orange Free State, and the Cape Provinces, particularly the Western Province. Natal and Swaziland hwe infected farms, losses being very heavy in both districts. Not only is the Union affected, but Southern Rhodesia has infected farms, as reported by Bevan, and the disease undoubtedly exists in both North-Eastern and North-Western Rhodesia. Sera sent from Livingstone have proved posi­tive when tested at the Laboratory, and there is also evidence indicating that it occurs in Northern Rhodesia. It is very probable that all parts of the continent of Africa into which imported cattle have been intro­duced contain infected cattle, as there is no restriction on the exporting of infected cattle from Europe, and no countries as yet require cattle to be subjected to serological tests before importing them.

During 1913, Mr G. N. Hall was successful in establishing the identity of the South African and European diseases. He obtained a pure culture of the B. abortus from the stomach of a fretus which had been obtained from a suspected farm, and he was able to prove its morphological and cultural identity with the European strains. Cultures were obtained from Stockman in England and Zwick in Germany. These European strains and a South African one were agglutinated to the same degree by a positive serum. He found that in the majority of cows which aborted the sera would agglutinate in a dilution of 1 to 800, less often I to 500, and rarely 1 to 400. The total number of cows tested-aborted and norma} -was about 200. When tested with a positive serum the South African strain was found to be agglutinated to a slightly higher degree than the European ones. The highest dilution in which a water-clear agglutination was obtained wi~h any serum was 1 to 2000. This work was done between May and August 1913, and on Mr Hall's departure the work was handed over to Mr Viljeon, who did a few further experiments. He fixed the standard at, or over, which a serum was to be considered positive at water-clear agglutination in a 1 to 100 dilution. This standard is believed to be higher than the one uses} by EI1fOpean workers, which is said to be agglutination in a 1 to 50 dilution. Tests were undertaken which confirmed the previous work on the comparison of the South African and European strains. Standard emulsions in '5 per cent. carbolised physiological water were found to agglutinate better when fresh than after they had been stored for fifteen days in the ice - chest. A few tests were undertaken to see whether sera which had been kept for at least a month, and even become contaminated, still retained their agglutinating power to the same degree. No preservative was added to the sera, and when tested they were found to have retained their titre which they had when fresh. This observation is in accordance with those of a great many observers, and

ABSTRACTS. 295

it is an accepted fact that the stability of agglutinins is very great. The work was handed over to Dr Veglia in November 1913, and he carried out numerous tests on sera from cattle in .the outlying districts. In July 1914, the author took over the work, since when numerous experiments have been done, and tests on the sera of upwards of 2000 cattle from the field have been undertaken. After trying several methods of collect­ing sera in the field, the present system, which has proved satisfactory, was adopted. It consists simply of sending out small sterilised bottles of 20 c.c. capacity, and having the blood collected in them. These bottles, if despatched immediately, generally arrive with the serum which has been expressed, quite clear. The bottles generally arrive within a day or two of filling, so that contamination with staining of the serum as a result is never marked. Occasionalfy the serum is not expressed from the clot, but this might occur with any method, and does not present a series problem. The existence of contagious abortion has now been proved in a great many districts of South Africa. It must originally have been imported into South Africa with European cattle, and infected cattle have probably been imported from time to time and are still coming in. Up to the present, no methods of immunising against the disease have been introduced into the Union of South Africa, and it is not pro­posed at present to attempt them. The reason is that at present the disease, although widespread, is not considered beyond control by means of quarantine, when its presence has been established on a farm. Immunisation will not be undertaken until other methods of control fail, and it is hoped that judicious quarantine measures will reduce the spread. The author thinks infection is probably spread as much by the bull on the large ranches in South Africa as by other methods. Inges­tion is still an important factor, but probably does not play such an important part as it does in dairy farms and places where the cattle are kept close together. Much difficulty has been experienced in determining by serological tests whether a bull is infected or not. Positive reactions have occasionally been obtained from bulls of infected herds, but negative results are the rule. The use of a bull from an infected herd for clean cows, even when giving a negative reaction, is not advisable, as it seems certain that many such bulls are infected in the prepuce only, not giving­cause to agglutinin-production in the body. Again, bulls which give a positive reaction are not necessarily infected in the prepuce, w that the question of infection in bulls is a difficult one to settle. The agglutina­tion tests have been used chiefly in the diagnosis of the disease, but the complement fixation test has been used as well as a control in a few doubtful cases.

Eradication of the Disease from a Herd by Testing and Separation.-An experiment was undertaken with the object of completely freeing an infected herd from contagious abortion by removing all cattle that showed positive reactions to the agglutination test, the sera of the whole herd being tested at certain intervals. Sixty-three cattle-cows, heifers, and a bull-from a herd in which contagious abortion had existed for some time, were sent to a clean farm, that is to say, one on which the disease did not exist, nor did it on any of the neighbouring farms. Previous to the removal in August 1915, the whole herd had given negative reactions, with one exception: a cow which had calved normally in 1913, and was not in calf on arrival on the new farm. The serum of this cow, No. 1148, had given a positive agglutination in January 1915 in a dilution of I to 200.

In August 19 I 5, the agglutination test of her serum gave a positive result in a 1 to 1000 dilution. This cow was accidentally included in

ABSTRACTS.

the herd, but was taken out at the first test on the clean farm. The first test of the whole herd on the clean farm was carried out in January 1916, and five reactors in a dilution of 1 to 100 or over were obtained. The numbers of these were 1148, 3103, 3080, 3335, and 3329. They were removed and isolated a fortnight after being bled for testing purposes. The subsequent history of these five cows is as follows:-

Cow No. 1I48.-Calved on 28th August 1916. The calf was big and -strong, and the fcetal membranes came away normally. This cow, never­theless, showed abortion bacilli in the milk, guinea pig inoculations giving positive results. She calved again on 26th December 19I7, in a normal manner, the milk again proving to be infected with abortion bacilli, as -shown by guinea-pig inoculation. The after-birth was infected as well as the milk at the second calving, and was probably infected at the first, but tests of it were not then made.

Cow No. 3103.-Aborted on IIth November 1915. The fcetus could not be found. Inoculation of the milk into guinea - pigs gave positive results, therefore the cow must have been harbouring abortion bacilli in the milk.

Cow No. 3335.-This cow aborted on 13th December 1917, two years -after isolation from the herd. She had been kept isolated with other reacting cows, and had not calved in the interval, although she had had -access to a bull. Her milk proved positive on inoculation into guinea-pigs.

Cow No. 3080.-Has not calved since separation from the herd. Cow No. 3329.-Calved in a normal manner, without retention of the

after-birth, on 25th February 1917, the calf being strong and active, and not small in size. The cow's milk, however, gave a positive result on inoculation into guinea-pigs. The after-birth was not obtained, so that it is impossible to give a definite opinion as to whether the uterus was affected -or not.

The second test of the complete herd two months later revealed the presence of three further reactors, Cows Nos. 3307, 3380, and 2737. These three were immedifltely isolated with the previous five in a separate camp on the farm to which none of the other cattle had access.

Cow No. 3307.-Has not calved since isolation. Cow No. 3380.-Calved normally on 4th November 1916. There was

no SuspiclOn of abortion, and the cow may be regarded as having calved in a normal manner. Guinea-pig inoculation of milk was not done in this -case.

Cow No. 2737.-Aborted in June 1916. The agglutination of this cow's serum was 1-2000 at the date of abortion. The fcetus was not obtained. The abortion occurred in the separate camp of the farm in which the cow had been isolated with the other reactors. The cow was quite dry on arrival at Onderstepoort a week after aborting, so that no milk could be obtained for guinea-pig inoculation.

If was decided in future to bring all the reacting cattle to Onderstepoort before calving had taken place, to ensure material for guinea-pig inoculation. All the reactors were brought up to this Laboratory for observation after the cow No. 2737 had aborted, and were no longer isolated on the farm itself as they previously had been.

The third test was made in May 1916 and there was only one positive reactor, Cow No. 3403.

Cow No. 3404.-Aborted on 22nd July 1916, the Bacillus abortus being obtained in pure culture from the milk. The fcetus was at about the seventh month of pregnancy and retention of the afterbirth occurred. The abortion occurred at Onderstepoort, the cow having been brought up in June 1916

ABSTRACTS. 297

with the other reactors. The cow again aborted on 14th December 1917, the fcetus this time being fully developed. Retention of the afterbirth again occurred, and the milk of the cow, emulsions of afterbirth, and stomach -contents of the fcetus proved infective for guinea-pigs.

The fourth test was carried out in July 1916, and two further reactors were (ound, cows Nos. 3201 and 3334, both of which were immediately brought .to Onderstepoort.

Cow No. 320I.-Calved apparently normally on 6th October 1916. The >calf was small and weak, but lived. B. abortus was obtained in pure :Culture from the milk of the cow. The calf was probably premature, but no retention of the afterbirth of the cow occurred.

Cow No. 3334.-Calved on 19th September 1916. The calf, though .alive when born, could not stand up or suck, and was very small though well formed. It appeared to be at about the seventh month of gestation, and was killed for culture making. No cultures of B. abortus were obtained from the calf, but were obtained from the milk of the cow. The cow again aborted on 16th August 1917, the fcetus this time being at the third or fourth month of gestation. Guinea-pig inoculations from the cow's milk, afterbirth, and the stomach contents of the fcetus proved positive.

The cows still remaining were all sent to another clean farm (Farm A). On 17th August 1916 the fifth test was made, and there were no further positive reactors. In this experiment a bull, No. 2600, was running with the herd of cows, but at no tIme did he give an agglutination of even I to 10. This does not exclude the possibility of his being infected at least to a slight degree in the prepuce, but as he had not served a cow which had aborted since August 1915, and only a few with positively agglutinating sera, 1t was presumed that he was probably not infected.

The herd from which the sixty-three cows and a bull were taken in August 1915 had been an infected one for a year previously. It had originally con­sisted of about 100 cows, heifers, bulls, and a few oxen. Ten actual abortions took place in it previous to AUll,ust 1915, and sixteen other cows were positive reactors to the agglutination test. The herd had been running on a farm (C), and in August 1915 the aborters and positive reactors were brought to Onderstepoort, the sixty-three cows and bull used in this experiment being those left over as non-reactors.

At the present date, two years after the last test of the herd after removal to Farm A, no further reactions have appeared, and none amongst the clean herd into which they were introduced.

The experiment shows that hy means of agglutination tests a herd can be cleaned from infected cattle if the tests be undertaken at definite mtervals and cows giving a pOSitive agglutination test are removed immediately.

Distribution of agglutinins in the different quarters of the udder.-A number -of tests were undertaken with the object of studying the agglutination titre of the milk from all four quarters of an infected cow. For these tests milk­whey was used and was obtained from the milk by the use of rennet. The ordinary concentrated essence of rennet was used in the proportion of 1 to 'So of milk. The milk was warmed to blood-heat before the rennet was added, and a solid coagulum formed almost immediately. A sufficiently dear whey was expressed from the coagulum within an hour or two. The whey thus produced was infinitely superior to any produced by the use of lactic acid, and had the advantage of being almost clear. The titre of the milk was found to vary when taken from the different quarters of the same cow, which observation has been made by many different workers. No definite ratio could be established between the agglutination titre of the cow's serum and that of the milk, as many factors influence the titre of the

ABSTRACTS.

milk-whey, such as the time of drawing the milk, i.e., much or little milk, and whether the cow has just calved or is nearly dry. In a few cases where a small quantity of milk was obained from a nearly dry udder the agglutina­tion titre of the milk was very high, even exceeding that of the cow's serum.

The milk-whey might be used as a medium through which contagious abortion could be diagnosed, but there does not seem to be any necessity for using it for this purpose except as a control to tests of the serum. Whatever result was obtained from the agglutination test of the milk, a test of the cow's serum would be necessary as well to control it, and a negative test of the milk would not justify a negative diagnosis. The first milk is not a very easy medium to use, as it is often thick and difficult to coagulate, and one would in any case have to bleed heifers.

In an article which appeared in the Journal of Agricultural Research: (7th February 1916, Vol. 1., No. 19) Cool edge suggests the use of the agglutination test as a means of testing a cow's milk for the presence of B. abortus. In his experiment no tests of the cow's sera are recorded and infection of the udder via the teat canals is assumed in almost every case. He inoculated 35 cc. of a young broth culture of B. abortus into the right quarter of a cow whose milk had always appeared in the quarter into which the culture had been inoculated, and soon after in the other three quarters. almost simultaneously. Cool edge assumes that infection spread from the infected quarter to the others during milking on the hands of the milker. Though this may not be impossible, one is inclined to think that the infection from the infected quarter first caused agglutinin production in the blood, and that from there the other quarters became possessed of agglutinins. The simultaneous appearance of agglutinins in the other three quarters also favours this view. No mention is made of the bacilli of con­tagious abortion havll1g appeared in the other quarter~. A record of another case in this article is more convincing. A cow which had never aborted was shown to have milk which would agglutinate weakly in all the quarters except the left fore. Guinea-pig inoculations with milk from each of the three positive quarters were positive, but the left fore quarter gave a negative result. Four months later the milk from the left fore quarter showed agglutinins, but at the date of publication the milk had not given :1,

positive result in guinea-pigs. The evidence here is incomplete, but is in agreement with the evidence obtained by other writers that local anti-body production to an organism can undoubtedly occur in the udder. Much importance is attached in this paper by Cool edge to infection of cows via the teat canaL Although it would be wrong to exclude this route, and it would appear to be supported by the evidence of Schroeder and Cotton that the only infected parts of a non-pregnant cow are the udder and supra­mammary glands, and it is known that the actinomyces can grow up the teat canal, yet it seems more likely that after the udder becomes infected' originally from the blood stream the bacilli set up a habitat there, and then infect the glands. Amongst the cows which have calved apparently normally here, but showed bacilli in the milk on the day of calving, it is certain that infection did not reach the udder from the soiled hands of the milker or from discharges soiling the udder. The cows were running on a large farm, and the chances of soiling of the udder from infected material were remote.

The Occurrence if the Bacilli in the Lymph Glands and other Organs of .Infected Animals.-These experiments were conducted with the object of confirming the statement of Schroeder and Cotton 1 that in the non-pregnant infected cow the udder and its lymphatic glands, and occasionally the pelvic

1 "Journal of Agricultural Research," Vol. 1., No.9, 4th April 1917.

ABSTRACTS. 299

glands, are the only portion of the body in which abortion bacilli are to be found. Four infected cows were used, all of which were reacting to the agglutination test, and their numbers were 3334, 943, 2939, and 3547·

Cow No. 3334.-Calved 19th September 1916; calf alive, and at about the seventh month of pregnancy. It eQuid not stand or suck, and was killed. The agglutination titre of the cow at the time of calving was I to 500, and she had been isolated as positive two months before. The atterbirth was retained, and the milk proved ineffective for guinea-pigs. The cow aborted on 16th August I9I7, the fcetus being at about the fourth to fifth month, retention of the afterbirth occurred, and the milk still proved to be infected, as were also both the afterbirth and the stomach contents of the fcetus. On 21st August 1917 she was killed, and at the date of death her agglutination titre was I to 500. Various parts were inoculated, but, unfortunately, although every care was taken, a large number of the guinea­pigs inoculated died of peritonitis, some as late as three weeks after inoculation. The cow had only two quarters to the udder, two of the ·quarters being congenitally absent, so milk was taken from each, and portions of each were used for inoculation. Milk was positive, portions of the right half of the udder were negative, both pelvic glands were positive, and no other positive results were obtained. The strong infec­tion of the pelvic glands is noteworthy, the cow having aborted five days previously.

Cow No. 943.-Aborted on 30th April 19 I 5, before arrival here. Aggluti­nation I to 200. Calved, apparently normally, on 20th September 1916. Agglutination of cow at birth of calf was I to 800. The afterbirth was not retained, but the cow suffered from metritis for a month after calving. The milk was not tested. Killed on 29th August 1917, on account of a -dislocated hip, and on post-mortem a normal fcetus was found in utero, with fcetal membranes normal in appearance. Agglutination of cow at date of -death was I to 100 to 1 to 200, fcetus negative. The cow was dry when killed, so no milk was inoculated. Of the guinea-pigs inoculated from this cow, one surviving from inoculation with the left forequarter of the udder was negative; both hindquarters were positive, although one guinea - pig from the left hind was negative, the other being positive. Both supra­mammary glands were positive, the pelvic glands negative, as were fluids from the thoracic duct and bile. Guinea - pigs inoculated with uterine material and stomach contents of the fcetus died of peritonitis.

Cow No. 2939.-Aborted 4th July 1915; afterbirth retained, and pure ·cultures of B. abortus were obtained from it. The agglutination titre of the cow at the date of abortion was 1 to 2000. The cow aborted again on 25th August 1916, the calf this time being fully developed, but born dead. The milk, afterbirth, and stomach contents of fcetus were all infected, as shown by guinea-pig inoculation. A third abortion occurred on 20th June 1917. The fcetus was again fully developed, and the afterbirth retained, and on inoculation into guinea-pigs, the afterbirth, stomach con­tents, and milk were again positive. On 6th September 19I7, an attempt was made to remove the udder of the cow, but although the operation was successful the animal did not survive it for more than a day, death being undoubtedly due to delayed chloroform poisoning. Various portions of the cow were used for guinea-pig inoculation; the udder, milk, supra­mammary glands, and gastric glands proved positive, though the result in the case of the gastric glands may probably be due to the cow having eaten a portion of her infected afterbirth at the abortion three months previously. Portions from all four quarters of the udder were mixed, and not inoculated separately.

300 ABSTRACTS.

Cow No. 3547.-Aborted in 1910. When first tested in 1914, her agglutination titre was I to 1000. This cow never calved again during the seven years after she aborted, and at time of death, which was due to traumatic pneumonia, the agglutination was only 1 to So. The uterus at post-mortem was not pregnant, and contained a quantity of yellowish­brown mucus. The results of guinea-pig inoculation from the cow are as, follows: The udder, the supramammary glands, and pelvic glands were posi­tive; all other parts negative.

The results obtained here correspond with those of Schroeder and Cotton, with the exception that one guinea-pig inoculated from gastric glands proved positive. With the exception of two guinea-pigs inoculated from the udder and supramammary glands of Cow No. 3547, and those from milk, afterbirth, and stomach contents, none of the inoculated guinea-pigs, has shown any definite lesions, and their agglutination titres have been taken as the indicators of infection. There seems little doubt that the udder and its lymphatic glands are the only infected parts of a non-pregnant infected cow, but how the udder is infected originally is not settled. Allow­ing for a small proportion of cases where infection originally occurred via' the teat canals, and this proportion must of necessity be small, particu­larly under ranching conditions, one is left with two possible channels, by which the bacillus may reach the udder. The one channel is via the blood stream direct to the udder after absorption from the alimentary tract or from the vaginal mucous membrane in infection via that channel. The other is by the pelvic glands to the supramammary glands, and thence to the udder from an infected uterus. Schroeder and Cotton have proved that by infecting the udder the uterus can become infected, but the reverse process probably occurs as often. Infection can apparently travel along lymph channels in the opposite direction to the lymph stream, and this undoubtedly occurs in tuberculosis of the udder. M'Fadyean considers that tuberculosis of the udder is usually the result of infection via the lymph stream from lesions in the abdominal cavity. It is not known whether it is necessary. for the udder to become infected before the uterus can, whatever the point of entry of infection, but further evidence on this point may be forthcoming. (E. M. Robinson, Fzfth and Sixth Reports qf the Director qf Veterinary Research,. Union of South Africa, 1918, pp. 337-372.)

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