Observations on Tuberculin Sensitivity in Cattle in Kenya

Brit. vet.]. (1965),

121,

319

OBSERVATIONS ON TUBERCULIN SENSITIVITY IN CATTLE IN KENYA By F. G.

WADDINGTON

Food and Agriculture Organization of the United

ations

SUMMARY

An investigation of tuberculin sensitivity in cattle in Kenya is still in progress. Temporary sensitization of cattle to tuberculin is of common occurrence. This may be due to infection with human type M. tuberculosis and also to atypical mycobacteria. True progressive bovine tuberculosis appears to be of low incidence. Cattle showing non.-specific reactions to tub erculin are found mostly in areas adj acent to the larger tracts of water and swamps. The isolation of psychrophyllic strains of mycobacteria from tuberculin sensitive cattle in such areas is recorded.

INTRODUCTION

For many years, tuberculosis was considered to be virtually non-existent among the cattle population of K enya and the importation of exotic stock has been carefully controlled by tuberculin testing. Minor outbreaks of the disease have been detected at infrequent intervals and controlled by test and slaughter but no systematic survey of incidence had been conducted. Meat inspection has been carried out by trained staff at the larger abattoirs and small slaughter houses and condemnations on account of tuberculosis have b een very rare. For example, at the Athi River abattoir of the K enya Meat Commission, where meat inspection is under the direct control of veterinarians, only I4 carcases were found to h ave tuberculous lesions in a total of 72,782 cattle slaughtered in I 96 I . Sporadic cases of tuberculosis in cattle have been detected at the time of m eat inspection of apparently healthy cattle, at post-mortem examination of cattle which had died from other causes and following tuberculin tests carried out for sale purposes or in connection with the investigation of infection d etected in carcases. There appears to be no history of clinical tuberculosis causing a progressive disease, loss of condition or mortality in cattle. No large scale survey by means of tuberculin testing has previously been undertaken; but the standard singJe intradermal comparative tuberculin test, applied to sale animals, or in the control of the minor outbreaks d escribed above, has frequently given results which have not been supported by the postmortem findings in those r eactors which have been slaughtered. "No visible E

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lesion" reactors have been noted from time to time; and in consequence, cattle failing the test have, more recently, been retested before a decision to slaughter has been taken and have been found to pass a later test. The epidemiology of tuberculosis in the human population of Kenya is relevant to the veterinary problem now being investigated. Tuberculosis was introduced comparatively recently, being unknown in up-country districts 50 years ago, and now there is a high incidence; the disease, in fact, is the most important public health problem in Kenya today. From information provided by the W.H.O. Tuberculosis Unit, it appears that among some 2,000 isolations of M. tuberculosis from Kenya patients in the past four years not a single culture was considered to be of the bovine type. It is most unlikely that this would be the case if bovine tuberculosis were established in the cattle population, as the African livestock owners live in close contact with their animals, milk is seldom boiled and meat is frequently eaten in only a half-cooked state. A little over three years ago, 14 of 50 Government-owned work oxen used on a rice-growing scheme were found to be infected with tuberculosis at slaughter. Fifty remaining oxen still working in the area were tuberculin tested and some reacted, though no lesions were found at post-mortem. It was felt that this might indicate that the disease was becoming more widespread than hitherto. In view of this an investigation was begun to ascertain the extent to which tuberculosis does in fact exist in the African-owned cattle in Kenya and to assess the reliability of the tuberculin test under East African conditions. The investigation is incomplete but a number of interesting features have emerged which seem worth recording at this stage, although further findings may lead to modification of the present views. METHODS

( I) Survey Tuberculin Testing Kenya has a population of nearly 6,000,000 cattle in African ownership and, for the purpose of assessing the incidence of reactors to tuberculin, the target of the survey was to tuberculin test 12,000 head of cattle in groups of 150 and 250 at various points scattered over the main cattle areas of the country. Though the total number of cattle tested in the survey will be only 0·2 per cent of the bovine population, it is considered that this will provide a reasonable assessment of the reactor rate, as by far the greater part of the cattle share communal grazing and the groups of animals tested are true random samples. The data recorded and discussed in the following sections have been obtained as a result of survey testing of 5,896 cattle in 14 districts of Kenya and from experimental tests of slaughter stock and of Government-owned cattle on two exp~rimental farms. When arranging to test cattle in a certain area, the local Chief or Headman is informed through the Administration and Veterinary Department that about 200 animals are required to be present at a crush site. Certain cattle owners are then asked to bring their animals along and, as nothing is known of the incidence of tuberculosis in their herds, randomization is assured. All the survey testing is carried out by the single intradermal comparative

TUBERCULIN SENSITIVITY IN KENYA CATTLE

321

method, as described by the Ministry of Agriculture, Fisheries and Food in Great Britain. The tuberculins used are obtained from Weybridge; mammalian (human) purified protein derivative (PPD) at 2'0 mg.jm!. and avian PPD at 0'5 mg.jml.; the dose of each being 0'1 m!. Interpretation of results is according to the standards laid down for use in Great Britain. The presence or absence of non-specific infection, as shown by the incidence of avian reactions, is taken into account when interpreting the test, and reactors .are classified as "failures" or "inconclusives". When making comparisons between the reactor rates in different areas, as recorded later in this article, failures only are considered. The sites of injection of the tuberculins are cleansed and demarcated by wet shaving, as clipping of the fine short hair of the Zebu animal has been found unsatisfactory. All cattle tested are identified by means of serially numbered plastic ear tags, inserted on the first day of the test.

(2) Experimental Tuberculin Testing In addition to the general survey, Government-owned cattle on two experimental farms run by the Veterinary Department have been used for critical testing with different dilutions and types of tuberculin and also to study changes in non-specific reactions over a period. This last point is discussed below but the findings with the different tuberculins are not recorded at this stage. On two occasions it has been possible to tuberculin test groups of cattle before slaughter, to examine the carcases of the reactors during meat inspection and to obtain material from them for cultural examination and typing of mycobacteria. Re-testing of failures and inconclusives detected during the survey testing has been done from time to time, to study changes in the reactions.

(3) Examination of Post-mortem Materi~l and Culture Typing A few specimens of tuberculous lesions detected at slaughter houses during the past year have been examined and submitted for laboratory investigation and so has material obtained from carcases of slaughter cattle which had been tuberculin tested. Full details of the specimens are not given in the text but the results of culture typing are included in the discussions which follow. The isolation and typing of acid-fast bacilli from post-mortem specimens have been done at the Veterinary Research Laboratory, Kabete; and also at the W.H.O. Tuberculosis Laboratory, Nairobi. (4) Collection of Relevant Population Data Details of cattle and human populations of the various districts of Kenya and the incidence of tuberculosis in the human population have been obtained from the Veterinary and Medical Departments. . RESULTS AND DISCUSSION

The total cattle population in the areas in which the 5,896 head were tested is

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2,524,000, so that 0-22 per cent of the cattle were tested, a close approximation to the sample target already mentioned_

( I) Survey Tuberculin Test Results in 14 Districts In many districts the herds are small, often comprising less than a dozen cattle_ In other districts where the herds are larger, several owners brought only part of their cattle to ensure that the sample was reasonably representative for the area_ The test results (Table I) show that tuberculin sensitivity is present in varying degrees in cattle in all the areas surveyed so far. Further investigation, by means of slaughter of reactors and cultural examination of post-mortem material, will be required to show conclusively the extent to which bovine tuberculosis is responsible for this sensitivity_ If the tuberculin reactions in cattle are accepted as indicating the presence of true bovine tuberculosis, it is reasonable to assume that this type of infection has also been recently intr~duced and that it would spread rapidly through the cattle population, becoming a progressive and fatal disease, especially bearing in mind that herds are intermingled in the Mrican areas and cattle are closely herded by day and sleep in close contact in their kraals at night_ These factors, coupled with the poor hygiene of Mrican farmsteads, the sharing of watering facilities, the stress of enzootic tick-borne disease and the varying degrees of malnutrition experienced by the cattle during the dry seasons, are all favourable to the establishment of widespread and active bovine tuberculosis_ However, as noted earlier, there appears to be no history of clinical tuberculosis causing loss of condition or mortality_ It may be that the relatively higher resistance of the Zebu breed of cattle to tuberculosis, compared with that of European breeds, has modified the disease picture_ The low incidence of tuberculous lesions seen at meat inspection has already been noted_ Pending more intensive cultural investigations, some indication of the significance of tuberculin sensitivity in cattle may be obtained from an examinaTABLE I RESULTS

District I. Meru 2_ Embu 3· Nyeri 4- Kiambu 5- Fort Hall 6. Samburu 7· Elgeyo Marakwet 8. Kwale 9· Kilifi 10. Kajiado Machakos II. 12. Kitui 13· N .F.D. 14· Mombasa Overall

Number of cattle tested 453 47 0 4 02 953 9 26 443 3 66 15 0 3 01 25 6 163 264 247 5 03 5,89 6

OF

SURVEY TUBERCULIN TESTING

Failed 16 17 19 63 46 7

%

%

0

3-49 3- 61 4.72 6-61 4-9 6 I-58 3.00 0 2-3 2 0 1-84 2·27 0.40 0

Inconclusive 19 18 34 95 85 41 25 4 36 19 24 12 6 12

3-90 3- 8 3 8-45 9-9 6 9- 18 9- 25 6-83 2-66 11-96 7-4 2 14-72 4·54 2.40 2.38

186

3. 15

43 0

7·21

II

0 7 0 3 6 I

TUBERCULIN SENSITIVITY IN KENYA CATTLE

tion of the frequency distribution of the tuberculin reactions. This has' been done in one area and the results are detailed below.

(2) Frequency Distribution of Reactions to Tuberculin The first five districts listed in Table I are the five administrative areas of the Central Province of Kenya and therefore constitute one large block of territory. Climatically the districts are very similar and are inhabited by one main tribe, the Kikuyu. Methods of animal husbandry are therefore very similar over the five districts. I t is seen from the table that the incidence of true mammalian reactors per cent in these districts is higher than in any of the others listed. A total of 3,204 cattle was survey tested in the five districts and the frequency distribution of reactions to mammalian tuberculin has been analysed. Avian reactions have not been illustrated, as these are rare in the Central Province, the total incidence of (A + M -) reactions being as low as 0 '95 per cent. The increases in skin measurements, at the sites of injection of mammalian tuberculin, shown by all the animals tested, have been extracted from the test records and the frequency distribution of the different sizes of reaction is presented in histogram form in Fig. I. The histogram to the left of Fig. I shows the percentages of reactions grouped as those showing increases of 0 to 2 mm. and then at I mm. steps. In the histogram to the right, minor errors in skin measurements have been adjusted by grouping measurements 0-2, 3-4 mm. and so on. The histograms are of course similar but that on the right has a smoother appearance. In an infected population, the reactions should show a bi-partite distribution, those indicating non-infected individuals being to the left and the infected to the right, of the reaction level taken as the criterion of infection; this being 4 mm. in the case of tuberculin reactions in cattle. Furthermore, the reactor component of the histogram for an infected population would normally show a range of size of reactions. No such bi-partite distribution is seen in Fig. I, in which the general appearance shows a predominance of smaller reactions causing a build-up of the left side of the histogram and a rapid fall away to a few larger reactions on the right. This shape is certainly not indicative of a population with esta blished infection and, indeed, ifno notice is taken of the 4 mm. mark as the standard of minimum positive reaction to tuberculin in cattle, the shape of the histogram is that of a populatioI! in which infection is not established. Accepting 4 mm. as the minimum positive reaction to tuberculin, it can be seen that the shape of the histogram in Fig. I is indicative of temporary sensitization. It is, in fact, the same pattern as that which was obtained with the frequency distribution of reactions to mammalian tuberculin in attested herds, with non-specific infection present, in Great Britain (Hebert & Paterson, 1955) . (3) Re-testing of Reactors detected by Survf!J Testing As the histogram of the Central Province reactions indicated that temporary

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sensitization to tuberculin might be taking place, it was decided that groups of cattle, classified as failures during survey testing, should be re-tested to ascertain if any fading of reactions took place. Six groups of failures were re-tested after an interval of three months. No avian reactions were noted at the original tests and the test readings were interpreted as if non-specific infection was not established and the same interpretation was used on re-testing. The results are summarized in Table II. Approximately 38 per cent of the animals were still failures at the re-test. In groups V and VI all the 17 cattle passed the second test. In the first four groups, though some of the failures still remained in that category, it was noted that the mean increase in skin measurements to mamma-

TUBERCULIN SENSITIVITY IN KENYA CATTLE

32 5

TABLE II RESULTS OF RE-TESTS OF FAILURES

Re-test results Group

Number of original failures

Failed

Inconclusive

Passed

41 12

22 4

II

8

3

I7

7

5 6

14

5

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8 9

38

101

6

7 8 3

26

37

lian tuberculin of all the animals tested was declining, as shown in Table III. It is apparent therefore that fading of reactions to mammalian tuberculin was taking place, which supports the impression of temporary sensitization of cattle to tuberculin given by the frequency distribution of reactions. TABLE III MEAN REA CTIONS TO AVIAN AND MAMMALIAN TUBERCULINS AT TEST AND RE~TEST ( MMo)

Groups Avian site original re-test Mammalian .rite original re-test

I

II

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8 °17 5" 2 9

At this stage of the discussion, it may be recorded that during the past year five cattle and one goat were found to have tuberculous lesions at meat inspection carried out in rural slaughter-houses in the area where the fading of reactions was demonstrated. Cultures from two of the specimens were too contaminated for further investigation, though acid-fast bacilli were presento The four pure cultures of Mycobacteria which were isolated were typed as: one atypical mycobacterium, growing at 20 °C (from the goat), and three human type tubercle. Both atypical and human strains would be likely to cause temporary sensitivity to tuberculin in cattle. (4) The Relationship between the Incidences of Positive Reactors to Mammalian Tuberculin in Cattle and T~berculosis in the Human Populations in the Same Environments

In view of the evidence of temporary sensitization of cattle to tuberculin, the possibility that this may be due to the transmission of human type tubercle from man to cattle merits consideration. The isolation of the three cultures of human type mycobacteria supports this possibilityo Furthermore, it should be noted that human sensitization of cattle is much more likely in Kenya than say in the United Kingdom because the chance of contact with infected people or contaminated material is much greater.

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The incidence of tuberculosis in the human population of the different districts in which cattle tuberculin tests have been carried out, the mammalian reactor incidence and also the ratios of people to cattle are shown in Table IV. The greater the number of people having possible contact with cattle the more likely is man to cattle infection. If human sensitization of cattle is occurring, the resultant reactions in cattle will be of mammalian type, so the failures only in the cattle t ests are presented for comparison with the human data. Inconclusives are not taken into account in this correlation because, while they may be due to developing or fading mammalian reactions, they may also be due to non-specific infection, a factor to be reviewed later in this article. TABLE IV COMPARISONS BETWEEN THE [N CIDENCES OF TEST FAILURES IN CATTLE AND HUMAN TUBERCULOS[S IN THE SAME AREAS

District I.

2. 3· 4· 5· 6. 7· 8. 9· 10. II.

[2. [3 · [4·

Meru Embu Nyeri Kiambu Fort Hall Samburu Elgeyo Marakwet Kwale Kilifi Kajiado Machakos Kitui N.F.D. Mombasa*

lru:ideru:e oj human T.B. per 100,000

Cattle tuberculin test Failures, %

Pulmonary

AllJorms

Ratios oj people to cattle

3'49 3·6 [ 4'7 2 6·6[ 4'9 6 1'5 8 3'00 0 2'3 2 0 [ ,84 2'27 0'40 0

110 1[2 149 [59 89 49 60 57 76 43 62 87 24 [49

115 12 9 170 177 96 54 75 58 78 76 64 88 25 [52

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·-See note in text

From Table IV it is seen that the percentage offailures, i.e. true m ammalian reactors, in cattle bears a direct relationship to the incidence of human tuberculosis, particularly to the pulmonary form. It is reasonable to assume that if the human population infected with tuberculosis is infecting the cattle and sensitizing them to tuberculin, the degree of threat will be modified by the ratios of the numbers of people to cattle. With this in mind, the relationship between human infection and sensitivity in cattle is even more apparent. The trend is more easily appreciated from the graphs, based on Table IV and presented in Figure 2. The points 1-14 on the graphs are the numbers corresponding to the districts listed in Table IV. It is seen from the graphs . that the rises and falls of Graph No. 2 are reflected in those of NO.3, with a major deviation at Point 14 which is explained below. The apparent effect of the ratio of people to cattle is seen when the three graphs are compared. For instance between Points 3 and 4, human tuberculosis shows a rise and a corresponding increase in the percentage

TUBERCULIN SENSITIVITY IN KENYA CATTLE

of failures in cattle is seen but this is much steeper. R eference to Graph No. I indicates a much higher ratio of people to cattle at Point 4 than at Point 3. This pattern is repeated along the graphs, with the exception of Point 14; which is interesting, as the cattle tested in Mombasa were permanently t ied up in open-sided byres at one place and confined to a fenced farm at the other. In both cases, these Mombasa cattle were isolated from the general population of the ar ea and had contact only with their attendants in the ratio of one man to t en cows or so. lt is suggested that the correlation between the trends of these graphs is more than coincidence and that human tuberculosis and tuberculin sensitivity in cattle are directly related. U..I

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BRITISH VETERINARY JOU R NAL, 121, 7

tuberculin were Mantoux t est ed. A comparison between the human Mantoux tests and the cattle tuberculin t ests is summarized in T able V and shown graphically in Figure 3. The results shown in T able V were obtained from a total of 1,273 cattle t ested in seven groups and a total of 315 people in contact. Many of the people TABLE V COMPARISONS OF CATTLE TUBERCULIN TESTS AND MANTOUX TESTS OF THE CATTLE OWNERS AND THEIR FAMILIES

Group

Cattle positive,

% I II III IV V VI VII

3·7 5.8 10·3 I 1'3 I I ·6 16 ·4 23 '4

Total number

1,2 73

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would not allow themselves to be Mantoux tested but it was possible to test a reasonable number in contact with the cattle groups; the numbers in human groups tested ranging from 9- 74 with a mean of 45 . The small group of nine people were the in-contacts with the smallest group of 27 cattle. The next lowest number of people was 25. As results are expressed as percentages, they have validity for comparative purposes and there is little doubt as to the correlation between the human and bovine results. The reactor rate in some of the cattle groups is very high but the fact that %
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TUBERCULIN SENSITIVITY IN KENYA CATTLE

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the Mantoux tests gave higher percentage positives in the human in-contacts in every case, seems to indicate man-to-cattle infection. In view of the high reactor rate in some of these cattle, a number of the reactors were retested, when it was found that reactions had faded considerably after three or four months. From three of the groups, i.e. II, VI and VII, a total of 67 cattle, all previous failures, were re-tested; the results being: 3 I fail, 16 inconclusive and 20 pass. Temporary sensitization was obviously occurring, for which human type tubercle could have been responsible.

(5) The Incidence of Non-Specific Reactions to the Tuberculin Test During the course of the survey testing, considerable variations have been observed in the incidences of reactions to avian tuberculin alone, (A + M - ) reactions, and of double positives to both tuberculins, (A + M +) reactions. It was noted that, in all the groups showing the higher incidence of nonspecific reactions, the cattle were grazing in the vicinity of permanent waters or swamps frequented with bird life, particularly herons and aquatic species. %

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Two of these groups were tested before slaughter and the reactors examined post mortem. In the first group of96 cattle there were five failures, if the test was interpreted on the basis that non-specific infection had not been established and one only on

33 0

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the basis that it had. All five animals were examined carefully post mortem but tuberculous lesions were found only in three of them, one having a primary focus in one lung, and the other two caseous mediastinal lymph glands . The lung lesion was in the one animal which was a failure with N.S .I. established and yielded a culture of human type tubercle. Cultures from the lesions in the other two animals proved to be atypical mycobacteria which grew at 20 ° C. In the second group of 247 cattle four animals were failures with N.S.I. not established but only one was still a failure when the test was interpreted with N .S.I. established. All four cattle were examined post mortem, together with a fifth in which the test was inconclusive but had shown pronounced reactions to both tuberculins. A careful search of the carcases and viscera failed to detect any lesions of tuberculosis. The mediastinal, bronchial and portal lymph glands were taken from each animal, for laboratory examination. Direct smears did not show any acid-fast bacilli and histological examination was negative. However, all five sets of glands yielded pure cultures of atypical mycobacteria, which grew well at 20°C. These findings suggest that atypical mycobacteria may have been responsible for sensitization of cattle to tuberculin and experimental sensitization of cattle with this type of culture will be undertaken. Meanwhile, it is significant that these organisms were isolated from cattle, in two widely separated areas, with high incidences of non-specific reactions to the test. The psychrophyllic nature of the organisms indicates that they are of saprophytic or cold-blooded origin; and two possible explanations for the prevalence of non-specific reactions at certain sites come to mind. The ecology of these areas may be more suitable for the survival of saprophytic mycobacteria on the grazing. The saprophytic acid-fast bacilli are, however, only rarely pathogenic and during the past year quite well developed tuberculous lesions in cattle have been shown to be due to atypical mycobacteria. Furthermore, there are considerable differences in the ecology of these areas; but a common factor in all cases is the proximity of waters supporting aquatic and wading birds. An alternative explanation is that birds feeding on fish and frogs from the nearby waters may be ingesting mycobacteria from these cold-blooded sources and spreading them on the grazing via their excreta. The findings of Darzino (1952) who records the isolation of atypical mycobacteria from persons in Brazil and also that edible frogs (Lepta dactylus pentadactylus) , naturally infected with similar organisms, were eaten locally, support this view. Attempts to isolate acid-fast bacilli from the intestinal contents of herons and from fish and frogs have been initiated. It may be noted that avian type tubercle has not been found in any material from bovine carcases. This type of infection has only once been confirmed in poultry in recent years in Kenya, although large numbers offowls are examined annually in the poultry diagnosis section of the Kabete Veterinary Research Laboratory. Further evidence that cattle constantly grazing near permanent waters may develop non-specific reactions to tuberculin was afforded by the results of tuberculin tests carried out on 96 head of slaughter cattle which had originated

TUBERCULIN SENSITIVITY IN KENYA CATTLE

33 1

from M asailand where it is unlikely that they would have spent any length of time grazing near any perman ent tract of water and which showed an increase offrom 14 to 23 per cent in non-specific reactions after grazing for ten weeks on a holding ground next to the permanent water of the Athi river. There was, however, no change in the state of true tuberculous infection as, with N.S.I. established, there was still only one failure, and the incidence of inconclusives remained at eight. With N.S.I. not established, the failures changed from seven to five, and inconclusives from 17 to 22. It would appear therefore that if the cattle were picking up non-specific infection in the area of the holding ground, the longer period spent in this environment would account for the increase in non-specific reactions. CONCLUSIONS

Sensitivity to tuberculin in cattle is wide-spread in Kenya, showing marked variations in degree in different districts; but the extent to which true bovine tuberculosis is responsible is not yet clear. Fading of reactions to mammalian tuberculin is common and the possibility that this may be due to temporary sensitization of cattle with human type tubercle is illustrated by the correlation between the incidences of tuberculin test failures in cattle and those of tuberculosis in the human populations in the same areas. Further evidence is provided by the isolation of human type tubercle from cattle examined post-mortem. Non-specific reactions to the tuberculin test are found to be most evident in cattle living in areas close to permanent waters frequented by birds which feed on fish and frogs. Atypical mycobacteria of a psychrophyllic nature have been isolated from tuberculin sensitive cattle in two such areas; and it is suggested that this type of organism is responsible for the non-specific reactions. ACKNOWLEDGEMENTS

The writer expresses his thanks to those concerned, on the staffs of the Veterinary Research Laboratory, Kabete and the W.H.O. Tuberculosis Laboratory, Nairobi, for their assistance in the isolation and typing of cultures. REFERENCES

HEBERT, C. N. & PATERSON, A. B. (1955). Vet. Rec., 67, 1143. DARZINO, E. (1952). Acta tuberc. Scand., 26, 170. Abstr. in Vet. Bull., 22,444.

(Received for publication November, 1964)