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Detection of the Spores of the Bacillus Anthracis in Soil
121
GENERAL ARTICLES.
SUMMARY. I. The thyroid gland of bull-dog calves IS not normal in histological structure. 2. The abnormality is not that which is associated with the general condition of cretinism, for the gland shows hyper- rather than hypo-functioning.
BIBLIOGRAPHY. (I) Emerson, C. P.; Osler and M'Crae: A System of Medicine, p. 683London, 19°9. (2) Leblanc: Camp. Rend. d. I. Soc. Bio!., 1902. (3) Apert: Ibid. (4) Regnault: Ibid. (5) Seligmann, C. G.: Journ. Path. and Bacterial., Vol. IX., 1904. (6) Sheather, A. L.: Journ. Camp. Pathol. and Ther., VA!. XXIV., 19I1. (7) Keith, A.: Tourn. of Anat. and Physiol., 3d ser., Vol. VIlL, 1913. (8) M'Carrison, R.: The Thyroid Gland. London, i917.
DETECTION OF THE SPORES OF THE BACILLUS ANTHRACIS IN SOIL.
By Captain R. H. KNOWLES, R.A.V.c., Veterinary Bacteriologist, Mesopotamia. THIS work was carried out at the Veterinary Bacteriological Laboratory, Baghdad, in connection with an extensive outbreak of anthrax at the Advanced Base Remount Depot, Baghdad. The outbreak commenced on 17th March 1921 and terminated on 21st April 1921. It involved the death of thirty-four horses and twelve mules. The usual symptoms of anthrax in equines were shown. There was a sudden rise of temperature up to 106 F., accelerated pulse, respiration hurried and laboured, profuse perspiration in many cases, and colicy pains, in most cases severe. Death took place in from one to two hours after the onset of the symptoms. Four of the cases showed moderate swelling of the throat, and one case showed a large swelling of the sheath. 0
Dz'agnosis. For the purpose of diagnosis the veins of the ear were opened under asceptic precautions. Smears were made for microscopic examination, and blood for cultural examination was taken on sterile swabs. This was done immediately after the death of the animal. Microscopic.-One blood smear was stained by aqueous solution of methylene blue (I per cent.), and another by Gram's method. They were examined, and if anthrax bacilli could not be discovered in the smears cultural examination was resorted to. Cultural.-The cultures were made on plain agar slopes, in wide test tubes (Ii inches in diameter), by sowing direct from the swab.
122
GENERAL ARTICLES.
A second tube of agar was inoculated from the first. The inoculated tubes were then incubated at 37° C. and examined at the eighteenth and twenty-fourth hours. The following table gives the result of microscopical and cultural examinations carried out as above : TABLE SHOWING METHOD OF DIAGNOSIS.
Lab. Number.
7026 702 7 70 59 7060 7062 706 3 7061 706 4 7 06 5 7066 706 7 7 068 7 06 9 7 0 70 7 0 71 70 72 7 0 73 70 74 7 0 75 70 76 70 77 70 78 7 080 7081 7 08 2 708 6 70 9 2 70 93 70 94 70 95 70 98 70 99 7 10 3 7 10 7 7112 7 IT 3 7I!4 7 108 7 10 9 7 11 5 7124 7 12 5 7126 7128 7 13 1 7 1 33
Date.
Description.
17.3. 21 do. 31.3. 21 1.4. 21 do. do 3.4. 21 do. do. do. do. do. do. do. do. do. do. 4.4. 21 do. do. do. do. do. do. do. :;.4. 21 8.4. 21 do. do. do. 9.4. 21 do. 11.4. 21 13-4. 21 do. do. do. 15.4 2 I do. do. do. do. 20.4. 21 do. do. 2I.4· 21
Bay Gelding do. Bay Gelding Mule Bay Gelding Bay Mare Mule. Bay Gelding Mule Bay Gelding Bay Mare. Bay Gelding Black Gelding Chestnut Gelding Bay Gelding Bay Mare. do. Brown Gelding Mule. Bay Gelding Mule Grey Gelding Bay Gelding Mule Chestnut Mare Bay Gelding Mule do. do. do. do. Bay Gelding Grey Gelding Bay Gelding do. do. do. Grey Mare. Bay Gelding Mule Bay Gelding Bay Mare . Bay Gelding Bay Gelding Mule Bay Mare. Chestnut Mare rlo. Bay Mare. Brown Gelding Bay Mare. do. Black Gelding Brown Gelding Chestnut Gelding Chestnut Mare Mule. Brown Gelding .
Microscopic Examination.
Cultural Examination.
+ + + + +
Not done. do. do. do. do.
+ + + + + + + + + + + + + + + + + + + + + +
Not done. do. do. do. do. do. do. do. do. do. do. do. do. do. do. do. do. do. do. do. do. do.
+ + + +
Not done. do. do. do.
+ + +
+
+ + +
+ +
-I
+ +
Not done. do. do.
+ + +
GENERAL ARTICLES.
12 3
Reference to the table will show that, out of forty-six animals proved to have died from anthrax, the bacillus anthracis was found : (a) Microscopically in thirty-four cases (74 per cent.). (b) Culturally in the twelve remaining cases (26 per cent.). That is, 26 per cent. of cases of equine anthrax failed to show anthrax bacilli in the blood by microscopic examination, and required a further examination by culture to detect the bacilli. If, however, the blood for examination was taken some time after death, it was found to be advisable to carry out inoculation tests in addition, owing to the possible presence of putrefactive organisms which would grow quicker than the Bacillus anthracis.
Source of the Infectt"on. The water supplied to the Remount Depot was not suspected to be the source, because it was obtained direct from the river Tigris. The forage used at the Remount Depot at this period came from two sources : 1. Grain and hay imported from India. 2. Green forage, consisting of lucerne and bersim, grown on the Remount Farm, adjoining the Remount Depot. The grain and hay imported from India has often been regarded with suspicion, as being the cause of i~olated cases of anthrax where no other source of infection appeared possible. Samples of grain and hay were therefore tested on 1st April 1921. Six samples of each taken from the consignments being used at this period were tested by cultural and biological methods, but no spores of the Bacillus anthracis were discovered. As the outbreak continued and appeared to be increasing in severity, it was decided to repeat the tests on the hay and grain, and in addition to test samples of green forage and also samples of soil from the plots on which the green forage was grown during the outbreak. A total of twenty-six samples were tested : (a) Three samples of hay. (b) Three samples of barley. (c) Two samples of lucerne and soil from each of plots Nos. 10, 26, and 27. Cd) Two samples of bersim and soil from each of plots Nos. 67 and 68. The plots are approximately 1200 square yards in extent, the farm being divided up into these plots by small irrigation channels. One pound of each sample was tested. This represented an extremely small proportion of the whole, so that the possibility of the detection of the spores of anthrax appeared to be small.
Laboratory Tecll1lt"que. Cultural and biological tests were carried out, although it was the latter test which was relied on, as it was shown to be more reliable than cultural methods by Glynn and Lewis. 1 1
"Journal of Hygiene," Vol. XII., No.2, June 1912.
124
GENERAL ARTICLES.
Preparation of the Material.-One pound of each sample to be tested was well agitated in flasks with a litre of sterile water. The resulting suspension was then filtered through very coarse gauze, and the filtered suspension was raised to 80° C. on a water bath and maintained at that temperature for ten minutes, in order to destroy any organisms in a non-sporulating condition. It was then allowed to stand for about two hours. Ten cc. of the lower part of the suspension was then pi petted off and used for sowing on agar and inoculation of guinea-pigs. "A" Cultural Examination.-·5 cc. of the suspension from each of the twenty-six samples was sown into about 20 cc. of plain agar at 40° C. A second tube of agar was inoculated from the first. Plates were made in large Petri dishes (6 in.) and incubated at 37° C. They were examined at the twelfth and eighteenth hours. No anthrax colonies were detected, and many of the plates were overgrown with other organisms, the majority of which appeared to be the Bacillus subtilis. "B" Biological E,ramination.-I cc. of the pipetted liquid from each of the twenty-six samples was inoculated into each of two guinea-pigs, subcutaneously on the abdominal wall. Both guinea-pigs inoculated with the suspension prepared from the soil of plot No 26 died, the first at the forty-fourth hour and the second at the fiftieth hour. The guinea-pigs inoculated with the material from the remaining twenty-five samples survived, and no abscesses developed at the seat of inoculation. Post-mortem examination of the two guinea-pigs revealed typical lesions of anthrax. In both cases there was an extensive gelatinous clear cedema around the seat of inoculation. The spleen was about twice the normal size and appeared softer than normal, and the liver showed considerable congestion. Microscopic examination showed the Bacillus anthracis to be present in large numbers in the subcutaneous cedema and in the spleen. The purple reaction with methylene blue was distinct in the case of the spleen. The bacilli were recovered in pure cultures in broth and on agar slopes. They gave typical growths on agar, and the bacillus was identified by microscopic examination of the cultures. Feeding with green forage at the Remount Depot was therefore stopped on 12th April 1921, and it will be seen from the table that the last case occurred on 21 st April 1921, that being the tenth day afterwards. A few days after the last case occurred feeding with green forage was re-commenced, except from plot No. 26 and the six plots surrounding it. No further cases of anthrax occurred. It would therefore appear that plot No. 26 was the source of the infection. The above tests only proved the presence of the spores of anthrax bacilli and not of the actual bacilli. The natural conditions existing at this period of the year (temperature'900 F. to 95° F. and moisture from irrigation) are, however, suitable for the growth of anthrax bacilli, and it is possible that a
GENERAL ARTICLES.
12 5
saprophytic existence is carried on. The infection may therefore have been by the bacillary form of the organisms. On the other hand, the results obtained from the cultural examinations appear to show that it is doubtful if the anthrax bacilli could compete with the more numerously present and more vigorously growing saprophytes. . Source of the Contamination of Plot No. 26. It is known that this area of land was used before the British occupation by the Turkish army for horse lines, and it is probable that the contamination is from a previous case of anthrax in the Turkish army, which quite possibly was skinned. Even if the carcase of an animal dead of anthrax was buried here it is possible that the spores could be carried to the surface by the agency of worms. SUMMARY. 1. The evidence appears to be fairly conclusive that plot No. 26 was the source of the cases, the actual infection taking place by contamination of the lucerne from the soil. 2. Although little hope of finding spores of the bacillus anthracis was held, the results would appear to indicate the employment of such tests in an outbreak of anthrax. 3. The results obtained in diagnosis indicate that the diagnosis of anthrax in equines should include cultural examination in cases where the bacillus cannot be detected microscopically. If the blood for examination is not obtained immediately after death, or obtained without asceptic precautions, the inoculation of guinea-pigs would be advisable.
BOVINE STAGGERS OR PUSHING DISEASE IN NATAL.
ISOLATION OF THE TOXIN OF MATRICARIA NIGELLMFOLIA, AND THE TREATMENT OF THE DISEASE. By Dr ROBERT SCHARRER, formerly Veterinary Research Officer, Pietermaritzburg. PUSHING disease, which is a well-known disease of cattle in Natal, is caused by a toxic plant, Matricaria nigell
GENERAL ARTICLES.
SUMMARY. I. The thyroid gland of bull-dog calves IS not normal in histological structure. 2. The abnormality is not that which is associated with the general condition of cretinism, for the gland shows hyper- rather than hypo-functioning.
BIBLIOGRAPHY. (I) Emerson, C. P.; Osler and M'Crae: A System of Medicine, p. 683London, 19°9. (2) Leblanc: Camp. Rend. d. I. Soc. Bio!., 1902. (3) Apert: Ibid. (4) Regnault: Ibid. (5) Seligmann, C. G.: Journ. Path. and Bacterial., Vol. IX., 1904. (6) Sheather, A. L.: Journ. Camp. Pathol. and Ther., VA!. XXIV., 19I1. (7) Keith, A.: Tourn. of Anat. and Physiol., 3d ser., Vol. VIlL, 1913. (8) M'Carrison, R.: The Thyroid Gland. London, i917.
DETECTION OF THE SPORES OF THE BACILLUS ANTHRACIS IN SOIL.
By Captain R. H. KNOWLES, R.A.V.c., Veterinary Bacteriologist, Mesopotamia. THIS work was carried out at the Veterinary Bacteriological Laboratory, Baghdad, in connection with an extensive outbreak of anthrax at the Advanced Base Remount Depot, Baghdad. The outbreak commenced on 17th March 1921 and terminated on 21st April 1921. It involved the death of thirty-four horses and twelve mules. The usual symptoms of anthrax in equines were shown. There was a sudden rise of temperature up to 106 F., accelerated pulse, respiration hurried and laboured, profuse perspiration in many cases, and colicy pains, in most cases severe. Death took place in from one to two hours after the onset of the symptoms. Four of the cases showed moderate swelling of the throat, and one case showed a large swelling of the sheath. 0
Dz'agnosis. For the purpose of diagnosis the veins of the ear were opened under asceptic precautions. Smears were made for microscopic examination, and blood for cultural examination was taken on sterile swabs. This was done immediately after the death of the animal. Microscopic.-One blood smear was stained by aqueous solution of methylene blue (I per cent.), and another by Gram's method. They were examined, and if anthrax bacilli could not be discovered in the smears cultural examination was resorted to. Cultural.-The cultures were made on plain agar slopes, in wide test tubes (Ii inches in diameter), by sowing direct from the swab.
122
GENERAL ARTICLES.
A second tube of agar was inoculated from the first. The inoculated tubes were then incubated at 37° C. and examined at the eighteenth and twenty-fourth hours. The following table gives the result of microscopical and cultural examinations carried out as above : TABLE SHOWING METHOD OF DIAGNOSIS.
Lab. Number.
7026 702 7 70 59 7060 7062 706 3 7061 706 4 7 06 5 7066 706 7 7 068 7 06 9 7 0 70 7 0 71 70 72 7 0 73 70 74 7 0 75 70 76 70 77 70 78 7 080 7081 7 08 2 708 6 70 9 2 70 93 70 94 70 95 70 98 70 99 7 10 3 7 10 7 7112 7 IT 3 7I!4 7 108 7 10 9 7 11 5 7124 7 12 5 7126 7128 7 13 1 7 1 33
Date.
Description.
17.3. 21 do. 31.3. 21 1.4. 21 do. do 3.4. 21 do. do. do. do. do. do. do. do. do. do. 4.4. 21 do. do. do. do. do. do. do. :;.4. 21 8.4. 21 do. do. do. 9.4. 21 do. 11.4. 21 13-4. 21 do. do. do. 15.4 2 I do. do. do. do. 20.4. 21 do. do. 2I.4· 21
Bay Gelding do. Bay Gelding Mule Bay Gelding Bay Mare Mule. Bay Gelding Mule Bay Gelding Bay Mare. Bay Gelding Black Gelding Chestnut Gelding Bay Gelding Bay Mare. do. Brown Gelding Mule. Bay Gelding Mule Grey Gelding Bay Gelding Mule Chestnut Mare Bay Gelding Mule do. do. do. do. Bay Gelding Grey Gelding Bay Gelding do. do. do. Grey Mare. Bay Gelding Mule Bay Gelding Bay Mare . Bay Gelding Bay Gelding Mule Bay Mare. Chestnut Mare rlo. Bay Mare. Brown Gelding Bay Mare. do. Black Gelding Brown Gelding Chestnut Gelding Chestnut Mare Mule. Brown Gelding .
Microscopic Examination.
Cultural Examination.
+ + + + +
Not done. do. do. do. do.
+ + + + + + + + + + + + + + + + + + + + + +
Not done. do. do. do. do. do. do. do. do. do. do. do. do. do. do. do. do. do. do. do. do. do.
+ + + +
Not done. do. do. do.
+ + +
+
+ + +
+ +
-I
+ +
Not done. do. do.
+ + +
GENERAL ARTICLES.
12 3
Reference to the table will show that, out of forty-six animals proved to have died from anthrax, the bacillus anthracis was found : (a) Microscopically in thirty-four cases (74 per cent.). (b) Culturally in the twelve remaining cases (26 per cent.). That is, 26 per cent. of cases of equine anthrax failed to show anthrax bacilli in the blood by microscopic examination, and required a further examination by culture to detect the bacilli. If, however, the blood for examination was taken some time after death, it was found to be advisable to carry out inoculation tests in addition, owing to the possible presence of putrefactive organisms which would grow quicker than the Bacillus anthracis.
Source of the Infectt"on. The water supplied to the Remount Depot was not suspected to be the source, because it was obtained direct from the river Tigris. The forage used at the Remount Depot at this period came from two sources : 1. Grain and hay imported from India. 2. Green forage, consisting of lucerne and bersim, grown on the Remount Farm, adjoining the Remount Depot. The grain and hay imported from India has often been regarded with suspicion, as being the cause of i~olated cases of anthrax where no other source of infection appeared possible. Samples of grain and hay were therefore tested on 1st April 1921. Six samples of each taken from the consignments being used at this period were tested by cultural and biological methods, but no spores of the Bacillus anthracis were discovered. As the outbreak continued and appeared to be increasing in severity, it was decided to repeat the tests on the hay and grain, and in addition to test samples of green forage and also samples of soil from the plots on which the green forage was grown during the outbreak. A total of twenty-six samples were tested : (a) Three samples of hay. (b) Three samples of barley. (c) Two samples of lucerne and soil from each of plots Nos. 10, 26, and 27. Cd) Two samples of bersim and soil from each of plots Nos. 67 and 68. The plots are approximately 1200 square yards in extent, the farm being divided up into these plots by small irrigation channels. One pound of each sample was tested. This represented an extremely small proportion of the whole, so that the possibility of the detection of the spores of anthrax appeared to be small.
Laboratory Tecll1lt"que. Cultural and biological tests were carried out, although it was the latter test which was relied on, as it was shown to be more reliable than cultural methods by Glynn and Lewis. 1 1
"Journal of Hygiene," Vol. XII., No.2, June 1912.
124
GENERAL ARTICLES.
Preparation of the Material.-One pound of each sample to be tested was well agitated in flasks with a litre of sterile water. The resulting suspension was then filtered through very coarse gauze, and the filtered suspension was raised to 80° C. on a water bath and maintained at that temperature for ten minutes, in order to destroy any organisms in a non-sporulating condition. It was then allowed to stand for about two hours. Ten cc. of the lower part of the suspension was then pi petted off and used for sowing on agar and inoculation of guinea-pigs. "A" Cultural Examination.-·5 cc. of the suspension from each of the twenty-six samples was sown into about 20 cc. of plain agar at 40° C. A second tube of agar was inoculated from the first. Plates were made in large Petri dishes (6 in.) and incubated at 37° C. They were examined at the twelfth and eighteenth hours. No anthrax colonies were detected, and many of the plates were overgrown with other organisms, the majority of which appeared to be the Bacillus subtilis. "B" Biological E,ramination.-I cc. of the pipetted liquid from each of the twenty-six samples was inoculated into each of two guinea-pigs, subcutaneously on the abdominal wall. Both guinea-pigs inoculated with the suspension prepared from the soil of plot No 26 died, the first at the forty-fourth hour and the second at the fiftieth hour. The guinea-pigs inoculated with the material from the remaining twenty-five samples survived, and no abscesses developed at the seat of inoculation. Post-mortem examination of the two guinea-pigs revealed typical lesions of anthrax. In both cases there was an extensive gelatinous clear cedema around the seat of inoculation. The spleen was about twice the normal size and appeared softer than normal, and the liver showed considerable congestion. Microscopic examination showed the Bacillus anthracis to be present in large numbers in the subcutaneous cedema and in the spleen. The purple reaction with methylene blue was distinct in the case of the spleen. The bacilli were recovered in pure cultures in broth and on agar slopes. They gave typical growths on agar, and the bacillus was identified by microscopic examination of the cultures. Feeding with green forage at the Remount Depot was therefore stopped on 12th April 1921, and it will be seen from the table that the last case occurred on 21 st April 1921, that being the tenth day afterwards. A few days after the last case occurred feeding with green forage was re-commenced, except from plot No. 26 and the six plots surrounding it. No further cases of anthrax occurred. It would therefore appear that plot No. 26 was the source of the infection. The above tests only proved the presence of the spores of anthrax bacilli and not of the actual bacilli. The natural conditions existing at this period of the year (temperature'900 F. to 95° F. and moisture from irrigation) are, however, suitable for the growth of anthrax bacilli, and it is possible that a
GENERAL ARTICLES.
12 5
saprophytic existence is carried on. The infection may therefore have been by the bacillary form of the organisms. On the other hand, the results obtained from the cultural examinations appear to show that it is doubtful if the anthrax bacilli could compete with the more numerously present and more vigorously growing saprophytes. . Source of the Contamination of Plot No. 26. It is known that this area of land was used before the British occupation by the Turkish army for horse lines, and it is probable that the contamination is from a previous case of anthrax in the Turkish army, which quite possibly was skinned. Even if the carcase of an animal dead of anthrax was buried here it is possible that the spores could be carried to the surface by the agency of worms. SUMMARY. 1. The evidence appears to be fairly conclusive that plot No. 26 was the source of the cases, the actual infection taking place by contamination of the lucerne from the soil. 2. Although little hope of finding spores of the bacillus anthracis was held, the results would appear to indicate the employment of such tests in an outbreak of anthrax. 3. The results obtained in diagnosis indicate that the diagnosis of anthrax in equines should include cultural examination in cases where the bacillus cannot be detected microscopically. If the blood for examination is not obtained immediately after death, or obtained without asceptic precautions, the inoculation of guinea-pigs would be advisable.
BOVINE STAGGERS OR PUSHING DISEASE IN NATAL.
ISOLATION OF THE TOXIN OF MATRICARIA NIGELLMFOLIA, AND THE TREATMENT OF THE DISEASE. By Dr ROBERT SCHARRER, formerly Veterinary Research Officer, Pietermaritzburg. PUSHING disease, which is a well-known disease of cattle in Natal, is caused by a toxic plant, Matricaria nigell