ELSEVIER VeterinaryMicrobiology40 (1994) 53-63

Post mortem diagnosis of Mycobacterium bovis

infection in cattle

L.A. Comer
CSIRO, Division of Animal Health, Animal Health Research Laboratory, Private Bag No. 1, Parkville,
Vic. 3052, Australia
(Accepted 6 January 1994)

Abstract

A tentative diagnosis of bovine tuberculosis can be made following the macroscopic detection at
necropsy of typical lesions. Histo-pathological examination of the lesion may increase the confidence
of the diagnosis but bacteriological isolation ofMycobacterium bovis from the lesion is the only way
to make a definitive diagnosis. The sensitivity of gross post mortem examination is affected by the
method employed and the anatomical sites examined. Careful examination of as few as 6 pairs of
lymph nodes, the lungs and the mesenteric lymph nodes can result in 95% of cattle with macroscopic
lesions being identified. Although during post mortem inspectionof carcases at abattoir all the principle
sites where lesions are to be found were examined, this procedure was found to be insensitive for the
detection of lesions. To determine the significance of cattle that give a positive reaction in diagnostic
tests but do not have visible lesions (NVL), a bacteriological examination is necessary. NVLs may
be due to early infection, poor necropsy technique or infection with mycobacteria other than M. bovis.
M. boris was found to survive best in frozen tissue and the tissue preservative, sodium tetraborate,
was found to have adverse effects on viability. It was found desirable to use two different culture
media for the primary isolation of M. bovis; agar media for rapid growth and egg media for control
of contamination. Additional control of contamination was achieved without adversely affecting the
viability by treating the specimen before culture with 0.075% hexadecylpyridinium chloride. The
addition of CO2 to the incubation atmosphere did not enhance the recovery ofM. bovis. Conventional
identification of isolates of M. bovis is by biochemical tests and cultural characteristics, but methods
employing monoclonal antibodies and DNA probes may be used to obtain a rapid identification.

Key words: Mycobacterium bovis; Cattle;Tuberculosis / bovine;Diagnosis

1. Introduction

Post mortem examination of cattle and bacteriological examination of lesions are critical
steps in the diagnosis of bovine tuberculosis. A tentative diagnosis of bovine tuberculosis

0378-1135/94/$07.00 1994 ElsevierScienceB.V. All rights reserved

SSDI0378-1135 (94)00008-K
54 L.A. Corner / Veterinary Microbiology 40 (1994) 53-63

can be made following the finding of typical lesions during necropsy. These lesions should
be collected for laboratory confirmation of Mycobacterium boris infection. A presumptive
diagnosis can be made using histopathology but a definitive diagnosis is dependent on the
isolation of M. boris. A major problem in the diagnosis of bovine tuberculosis is the long
time required to obtain a definitive diagnosis, that is, the isolation and identification of M.
boris. More than 12 weeks may be required for cultural confirmation of infection. In many
laboratories, histopathology is used to circumvent the long delay in making a bacteriological
diagnosis. In high prevalence areas a histological diagnosis may be satisfactory but it cannot
be relied upon in all situations. Improvements in diagnostic procedures which make them
more accurate or rapid can have considerable impact on disease control.
In the early stages of tuberculosis control programs, when the disease prevalence is high,
great reliance can be placed on diagnosis of tuberculosis based on detection of macroscopic
lesions during gross post mortem examination. Cultural examinations are rarely required
when the disease frequency is high and the cost of a misdiagnosis is negligible. When the
disease prevalence is low, as during the latter stages of an eradication campaign, the need
for a definitive diagnosis becomes more important and routine culturing of all suspected
lesions should be performed. Towards the end of a eradication campaign, as has been the
case in Australia during the last 10 years, it becomes increasingly important to determine
the true status of tuberculin reactors, especially where macroscopic lesions cannot be found
(the so-called non-visible lesion (NVL) reactor). In the later stages of an eradication
campaign exhaustive cultural examination of tissues from such animals may be necessary
to clarify their status.
In this paper, recent changes in two significant areas in the diagnosis of bovine tubercu-
losis will be reviewed. Firstly, the detection of infection through necropsy techniques,
including the collection of tissues from NVL reactors, and an examination of the sensitivity
of lesion detection by abattoir inspection procedures. Secondly, an examination of some of
the recent changes in bacteriological procedures for the primary isolation and the identifi-
cation of M. boris. The critical areas of consideration in the design of trials for the evaluation
of diagnostic tests for bovine tuberculosis will also be addressed.

2. Necropsy

The necropsy procedure employed to examine cattle that are reactors to a tuberculin test
can take one of two forms: an examination of tissues for macroscopic lesions, done at the
time of the necropsy, or, the aseptic collection of intact tissues (lymph nodes) for later
detailed examination in a laboratory. In the former procedure the lymph nodes are sliced in
situ and the cut surfaces examined for lesions. This procedure is employed when the
detection of gross lesions is sufficient, such as when bovine tuberculosis is endemic and the
prevalence of disease is high. Infection may then be confirmed by culture of the lesions.
The second procedure, the detailed examination, is usually performed in association with
the collection of tissues for bacteriological examination, hence the need for aseptic precau-
tions and the associated laboratory examination. The aim of aseptic collection is to prevent
or minimise the contamination of tissues with soil and faeces that may contain atypical
mycobacteria or other micro-organisms that could overgrow and obscure M. boris during
 L.A. Corner/ Veterinary Microbiology40 (1994) 53-63 55

culture and thus confuse diagnosis. L y m p h nodes are not sliced, but are dissected free from
surrounding fat with the capsule of the lymph nodes left intact to act as a physical barrier
to contamination. This second procedure is employed where the results of the necropsy has
significance in the subsequent treatment o f the herd or region. Such a situation might be
where a single tuberculin reactor is found in a previously uninfected herd or region, the
reactor is from a herd that is in the final stages of eradication, where there is insufficient
history on the herd, or where the cause of the tuberculin sensitivity is thought to be non-
specific, that is, due to causes other than M. boris.
The necropsy examination for the detection of lesions of bovine tuberculosis must be
thorough and all tissues listed in Table 1 should be identified and examined. A reactor in
which no visible lesions of tuberculosis are detected cannot legitimately be called a false-
positive or an N V L reactor unless all the tissues listed have been thoroughly examined.
Under the conditions of cattle husbandry in Australia, most tuberculosis infections in
cattle are acquired by inhalation (Lepper and Pearson, 1973; Corner et al., 1990). Between
70% and 90% of lesions are found in either the lymph nodes of the head or in the thoracic
cavity. However, because tuberculosis is a disease affecting the reticuloendothelial system,
lesions may occur in lymph nodes in virtually any anatomical region of the body, hence the
wide range of lymph nodes that need to be examined.
In order to optimise the post mortem examination procedure, we determined the distri-
bution of lesions in 374 tuberculous cattle. The lesions were detected using a detailed
necropsy procedure where all the tissues listed in Table 1 were examined.

Table 1
Tissues to be examined for lesions in cattle reacting to a tuberculin test

Head: mandibular ln*-left and right

parotid In-left and right
medial retropharyngeal In-left and fight
lateral retropharyngeal (atlantal) In-left and right
tonsils
Thorax: mediastinal In-anterior and posterior
tracheobronchial (bronchial) In-left, fight, cranial and medial
lungs
Abdomen: liver
hepatic In
spleen
mesenteric ln's along the entire length of the gastro- intestinal tract
kidneys
Carcase: caudal cervical (prescapular) ln-left and right
subiliac (prefemoral) ln-left and right
internal iliac In-left and right
medial iliac In-right and left
lateral iliac (deep inguinal) ln-right and left
gluteal (ischiatic) ln-left and right
sacral ln-left and right
superficial inguinal (supramaInmaryor scrotal) In-left and right
udder or scrotal contents and seminal vesicles
*In-lymph node.
Ref. Comer et al., 1990.
56 L.A. Corner/Veterinary Microbiology 40 (1994) 53~3

Table 2
The location of the most frequently affected tissues in cattle with single lesions of tuberculosis

Tissue % of lesioned cattle detected

medial retropharyngeal In (left and right) 29.4

mediastinal In (anterior and posterior) 28.2
bronchial In (left and right ) 18.0
lung 9.8
mesenteric In 2.9
parotid In (left and right) 2.4
caudal cervical In (left and right) 2.4
superficialinguinal (left and right) 1.2
Proportion of all cattle with single lesions
that were detected 94.3
Ref. Comer et al., 1990.

From the data, we ascertained which were the essential tissues to examine in order to
identify all the tuberculous cattle. In cattle with only a single lesion of tuberculosis, and
66% of tuberculous cattle had only one lesion, we found that 86% could be identified by
examination of only 3 pairs of lymph nodes (mediastinal, medial retropharyngeal and
bronchial) and the lungs (Table 2). Examination of 3 additional pairs of lymph nodes
(parotid, caudal cervical and superficial inguinal), and the mesenteric lymph nodes enabled
the detection of 95% of animals with lesions (Corner et al., 1990).
When lesions suspected to be tuberculous are detected they should be submitted for
bacteriological and histopathological confirmation. As more than one cause of granulomas
can occur simultaneously in an animal, up to three lesions from each animal should be
submitted for laboratory confirmation to ensure a correct diagnosis is made. If a lesion is
too small to divide, collection for bacteriology should be given preference.
Non-visible lesion tuberculin reactors can be encountered whenever cattle or other ani-
mals are tested. The lack of visible lesions may be due to a variety of reasons, for example,
visible lesions may be present and not observed, an animal may be in the early stages of
infection when lesions are too small to see with the naked eye or tuberculin sensitivity may
be due to infection with mycobacteria other than M. boris. The significance of NVL reactors
will vary with the history of the herd. In herds where cattle with lesions are found the NVL
reactors are of little significance. The real significance of NVL reactors is when no lesioned
reactors arc present in a herd.
N V L reactors should not be classed as " n o t infected" as many can be shown to be
infected with M. boris. The number found to be infected will vary with the prevalence of
M. boris in the cattle tested. The prevalence of culture positive NVLs can be as high as
10%. When lesions cannot be found in a tuberculin reactor the situation must be clarified
bacteriologically. The tissues that should be collected for culture are those most frequently
found to contain lesions (Corner et al., 1990; Table 2).

3. Comparison of abattoir and detailed necropsy for the detection of gross lesions
The degree of sensitivity of the necropsy procedure for the detection of lesions depends
on the time and diligence of the people conducting the examination. During abattoir post
 LA. Comer/Veterinary Microbiology 40 (1994) 53-63 57

mortem inspection, the principle interest of the inspectors is the suitability of the meat for
human consumption, not the detection of all diseased cattle. In Australia, where vigorous
post mortem inspection procedures are employed, abattoir monitoring should, ideally, detect
95% of lesions present in a carcase because the appropriate tissues are examined. We
compared the frequency and distribution of tuberculous lesions in 113 tuberculin reactor
cattle all taken from one tuberculous herd and randomly allocated to one of two methods
of examination. 25 were examined by a detailed post mortem procedure where the tonsils,
lungs, liver, spleen and kidneys were examined at post mortem and the lymph nodes listed
on Table 1 were collected at the time of post mortem for later examination for macroscopic
lesions in a laboratory. Only three mesenteric nodes, selected at random from the small
intestine, were collected for laboratory examination. The remaining 88 reactors were sub-
jected to routine abattoir post mortem inspection. If the two techniques were equivalent
there should have been equal proportions of cattle with lesions in each group. We found a
marked disparity between the results of the two treatments (Corner et al., 1990). The
abattoir post mortem inspection procedure detected an estimated 47% of lesions compared
to the number detected by the detailed post mortem procedure. The reason for the lower
detection rate in the abattoir can be related to the manner of examination. In abattoirs smaller
lesions may have been missed because there was limited time available for the examination
of each tissue and fine slicing of the lymph nodes in situ was very difficult. It was concluded
that abattoir inspection had low sensitivity but was a cost effective method of monitoring
large numbers of cattle for lesions of tuberculosis.

4. Storage of tissues

To obtain the best results from bacteriological examination of specimens, the specimens
must be presented to the laboratory in the best possible condition. In the pastoral area of
Australia necropsies are frequently performed large distances from the laboratory, maybe
as far as 1000 km, and often in hot weather (up to 40C). It has therefore been a routine
procedure in Australia to use sodium tetraborate (borax) to preserve the tissue and to control
surface contamination on tuberculous lesions collected for bacteriological examination. The
specimens were stored either in a saturated solution of borax or coated with a layer of
powdered borax.
We examined the effect of storage temperature ( - 20C and 6C) and the effect of borax,
on the viability of M. boris in tissues. Freezing was found to be the best way to preserve
M. boris in tissue. The change in viability of M. boris in the stored specimens was measured
by the change in the mean number of colony forming units per gram of tissue. In 25 tissues
stored at - 20C the number of viable bacteria decreased by an estimated 32% during the
course of 1 year (Fig. 1). In a second trial 10 tissues were stored at 6C with and without
the addition of borax powder. There was an 80% loss in viability in the first 3 days in the
presence of borax but only a 20% loss where borax was not used (Fig. 2). At the end of
the 56 day study in the second trial the loss in viability with and without borax was the
same.
The data therefore suggests the following strategies. For the detection of the greatest
number of viable M. boris in stored specimens, specimens should be chilled to 4C to 6C
58 L.A. Comer/Veterinary Microbiology 40 (1994) 53-63

Survival

100-

60 ....................................................

40 ...............................................

20 ...............................................

0 50 100 150 200 250 300 350 400

Time (days)

Fig. 1. Survival of Mycobacterium boris in tissue stored at -20C. Twenty-five tissues were each divided in to
40 pieces and the pieces randomly allocated to 5 pools. At each time of culture one pool was cultured and the
number of colony forming units (CFU) per gram of tissue was determined. The percentage survival of M. boris
was plotted against storage time.
Mean Count
450

40O
35O
3OO
25O
2OO
150
100

5O
0 i J i J i t F
0 5 10 15 20 25 30 35 40 45 50 55 60

Time (days)
Borate + No Borate

Fig. 2. The effect of the presence of sodium tetraborate (borax) on the survival of Mycobacterium boris in tissue
stored at 6C. Ten tissues were each divided in to 40 pieces. For each tissue the pieces were randomly allocated
to 10 pool samples. To one set of 5 pools the tissue pieces were covers with a thin layer of borax powder. At each
time of culture two pools, one treated with borax, were cultured and the number of colony forming units (CFU)
per gram of tissue was determined. The mean number of CFUs for the 10 tissues was plotted against storage time.

a n d c u l t u r e d w i t h i n 24 to 4 8 h o u r s o f collection. I f that c a n n o t b e d o n e the n e x t b e s t o p t i o n

is to f r e e z e t h e s p e c i m e n i m m e d i a t e l y u p o n collection. It is essential that s p e c i m e n s that are
stored f r o z e n r e m a i n frozen, e s p e c i a l l y d u r i n g a n y p e r i o d o f transport, until t h a w e d for
cultural e x a m i n a t i o n . W h e n facilities for f r e e z i n g are not available, c h i l l i n g for 4 8 to 72
h o u r s w i t h o u t b o r a x is p r e f e r a b l e to the u s e o f borax. T h i s c a n only b e d o n e i f the s p e c i m e n s
 LA. Corner/Veterinary Microbiology 40 (1994) 53-63 59

have been collected aseptically. Where gross contamination is present on specimens, borax
and chilling to 6C is required.

5. Bacteriology

The main factors that influence the success of primary isolation of M. boris from clinical
specimens are the culture media, the decontamination procedure and the incubation condi-
tions. Once an isolate is obtained a number of techniques can be employed to confirm the
identity of the isolate.

5.1. Culture media

Field strains of M. boris require enriched media for growth on primary isolation. The
media used are either egg based (e.g. Stonebrink' s medium (Lesslie, 1959) and Lowenstein-
Jensen medium with added pyruvate), or agar based and enriched with serum and/or blood
(e.g. modified Middlebrook 7H 11 medium (Gallagher and Horwill, 1977) and tuberculosis
blood agar medium, also called B83 (Cousins et al., 1989) ). The two basic medium types
have different attributes when used for the primary isolation of M. boris. In studies using
491 specimens collected from cattle that were tuberculin reactors, growth on agar medium
was found to be faster, with the mean time to the first appearance of recognisable colonies
being 27 days with B83 and 28 days with 7H11, compared to 36 days with Stonebrink's
medium. The faster growth rate was also reflected in the time taken for the maximum
number of colonies to appear. Again growth on B83 (mean 33 days) and on 7H11 (mean
35 days) was faster than on Stonebrink's (mean 42 days).
The advantages of the more rapid growth rate on the agar media were offset by the greater
susceptibility of these media to the growth of contaminants, even after the specimen has
been decontaminated. In a study using 362 clinical specimens, the agar medium (7H11 )
was contaminated twice as often as the egg medium (Stonebrink's) prior to decontamina-
tion, 8.3% compared to 4.7%. After decontamination with 2% w / v sodium hydroxide the
difference was more marked, 3.9% of specimens on the agar medium were contaminated
compared to 0.3% on the egg medium. For primary isolation, it is therefore recommended
that both egg based medium and an agar based medium be used for each specimen.

5.2. Decontamination procedures

Decontamination of specimens is frequently necessary to permit the isolation of M. boris

from contaminated specimens. If specimens are collected aseptically they may be cultured
without decontamination. The major problem encountered in selecting a reagent and the
concentration of the reagent for decontaminating specimens is the adverse effect most
reagents have on M. boris. The ideal reagent should have minimal toxicity for M. boris but
maximum toxicity for other, contaminating, organisms. As no ideal reagent has been found
the type and concentration of decontaminant used will be a compromise.
We examined the toxicity for M. boris of four commonly used decontaminants - hex-
adecylpyridinium chloride (HPC) at both 0.075% w / v and 0.75% w/v, benzalkonium
60 L.A. Corner~VeterinaryMicrobiology40 (1994)53-63

Table 3
Toxicityof fourdecontaminantsforMycobacteriumbovissuspendedas comparedto treatmentwithsteriledistilled
water

Decontaminant Concentration Percentageresidual

(% w/v) in viability
SDW*

HPC 0.075 73.0

0.75 51.5
Zepharin 0.25 48.5
Oxalic acid 5.0 42.7
NaOH 2.0 14.2
*SDW - sterile distilled water.

chloride (Zepharin) 0.25% w/v, oxalic acid 5.0% w / v and sodium hydroxide 2% w / v
(Table 3). We mixed a known number of viable M. boris colony forming units with sterile
tissue suspensions and then exposed the suspensions to the decontaminants for 30 min. We
measured the number of M. boris colony-forming units that survived the treatment with
these reagents (Table 3). There was great variation in the degree of toxicity. HPC was
found to be the least toxic, with the lower concentration, 0.075%, the least toxic of all. The
highest level of toxicity was observed with 2% NaOH, the traditional and the most com-
monly used decontaminant in mycobacteriology. The ability of 0.75% HPC and 2% NaOH
to control contamination was compared in 362 clinical specimens. Irrespective of the media
used, HPC gave complete control of contamination.
Overall HPC at the lower concentration (0.075%) was the superior reagent. HPC at
0.75% may be employed with individual specimens where HPC at 0.075% has failed to
control contamination. Although, of the reagents examined HPC 0.075% is recommended,
the selection of decontaminant and the concentration at which it is used may be varied,
subject to local conditions and the contaminants encountered.

5.3. Use of carbon dioxide

We found in a study using 43 clinical specimens that the inclusion of 5% C O 2 in the

incubation atmosphere had no effect on the growth rate or the number of colonies of M.
boris on primary isolation. However, CO2 concentrations in excess of 5% inhibited growth.

5.4. Laboratory animal inoculation

Laboratory animal inoculation for the primary isolation of M. boris is not recommended.
For contaminated specimens, where this procedure has been most widely employed in the
past, the culture methods outlined above are at least as sensitive as animal inoculation.

6. Identification

Identification of M. boris isolates has traditionally relied upon colony morphology and
staining characteristics, along with biochemical tests. More recently rapid techniques using
 L.A. Corner/VeterinaryMicrobiology40 (1994)53-63 61

monoclonal antibodies and DNA technology have been developed. M. boris are slow
growing, and on subculture require a minimum of 14 days for colonies to become visible
on media. On egg media the typical colony is small, rounded, pale yellow to buff with
irregular edges and a granular surface. On agar medium they are white, thin, rough and flat
with a central mound. Mycobacteria are described as 'acid-fast' because when stained with
hot carbol-fuchsin they resist decolorisation with acid-alcohol. When so stained M. boris
appear as red coccobacilli or short rods, 0.3-0.6/~m by 1-4/zm. In tissue sections and
lesion smears they are slightly longer.
There are a number of biochemical tests for M. boris (Vestal et al., 1975; Kent and
Kubica, 1985), the most useful being susceptibility to 2-thiophene carboxylic hydrazide
(TCH) and to isoniazid. These biochemical tests take up to 3 to 4 weeks to complete.
Conventional identification techniques require good growth on artificial media and take
from 3 to 4 weeks to complete following primary isolation. Recently, the rapid identification
of M. boris isolates has been facilitated by the development of immunological (e.g. mono-
clonal antibodies; Corner et al., 1988) and DNA probes (For discussion of the use of DNA
probes and polymerase chain reaction see Collins et al., 1994). These technologies allow
for the rapid identification of members of the Mycobacterium tuberculosis complex, which
includes M. boris, in less than a day, but isolates still need to be subjected to conventional
biochemical tests to confirm their identity at the species level.
A monoclonal antibody, 4C3/17 (Corner et al., 1988), has been found to have a high
specificity for M. boris. This antibody recognised all 156 strains of M. boris used in the
study and also some other members of the M. tuberculosis complex (6 of 7 strains of BCG
and 12 of 14 strains of M. tuberculosis). None of the other species of mycobacteria or other
genera examined bound the antibody, demonstrating a 100% specificity for the M. tuber-
culosis complex. A convenient dot blot technique (Veerman et al., 1990) using this mono-
clonal antibody for the identification of M. boris has been developed. Identification can be
made from a large colony or from bacterial growth scraped off a primary isolation slope.
The results of the identification are available in 2 to 3 hours.

7. Field evaluation of tests for bovine tuberculosis

In order to validly determine levels of sensitivity and specificity for a diagnostic tests,
accurate determination of the infection status of all animals used in the evaluation is essential.
The procedure used to identify the diseased animals must be independent of the test being
evaluated. For example, in evaluating diagnostic tests for bovine tuberculosis it would not
be appropriate to evaluate the interferon-gamma (IFN-y) assay (Rothel et al., 1990) using
as the criterion for disease diagnosis the results of a skin test, that is, based on another test
of the cellular immune system. For tuberculosis the only alternative to a test for cellular
immunity, that is sufficiently sensitive, is bacteriological examination. The definitive diag-
nosis of bovine tuberculosis is the isolation of M. boris. This is the best "Gold standard"
to use in the evaluation of any diagnostic test. For the evaluation of the IFN-y assay Wood
et al. ( 1991, 1992) used bacteriological confirmation of infection as the criterion for disease,
that is, as the independent assessment of disease status. There are 4 critical points to keep
in mind when designing trials to evaluate diagnostic tests:
62 L.A. Corner/VeterinaryMicrobiology 40 (1994) 53-63

1. The animals for inclusion in the trials must be selected at random. Thus to avoid bias
whole herds should be used or the animals can be selected at random from the population
under study.
2. All animals should be tested at the one time to avoid the possibility of one test interfering
with another test if two tests are being compared in the same animal. This was of particular
concern in the trials to evaluate the IFN-T assay as the skin test was known to affect the
titres in the IFN-T assay (Rothel et al., 1992).
3. All reactors to the test(s) being evaluated should be subjected to a detailed post mortem
and bacteriological examination. As a minimum, the three most frequently infected tissues,
that is, the mediastinal, bronchial and medial retropharyngeal lymph nodes, should be
collected, using an aseptic technique, for bacteriological examination. All the tissues listed
in Table 1 that are not collected for culture should be sliced in situ and examined for lesions.
4. All non-reactor cattle should also be examined post mortem. For practical and economic
reasons this may best be accomplished by passing them through an abattoir and subjecting
them to post mortem abattoir inspection. If the inspection procedure that is employed can
be enhanced by, for example, allowing more time for the examination of each carcase and
increasing the range of tissues examined to those nominated by Corner et al. (1990), the
efficiency of the procedure should approach an acceptable level of sensitivity.
Many of the trials designed to evaluate diagnostic tests for tuberculosis have not been
properly conducted and it is now difficult to draw valid conclusions from them. In these
trials invalid results were obtained because one or more of the following procedures were
used:
a. there was interaction between tests, for example, several different skin tests were done
on the one animal at the one time (Francis et al., 1978);
b. the animals were selected from the population by applying another test first (Lepper et
al., 1979);
c. in many trials the disease status of the reactors was based only on the presence of lesions
detected using insensitive procedures, for example, using routine abattoir meat inspection
procedures for the examination of reactors; and,
d. not all the cattle used in the trials were examined post mortem, that is, non-reactor cattle
were not killed or were not examined at slaughter (Francis et al., 1978).

8. Conclusion

Traditional methods of culture and post mortem examination are very effective procedures
for diagnosing tuberculosis. However, the efficacy of these procedures should not be taken
for granted. During the eradication program in Australia courses were run for field veteri-
narians to refine their skills for detailed post mortem and aseptic collection of specimens
for culture. Similarly, M. boris is a difficult bacterium to isolate and identify. A national
quality control program was established during the Australian campaign to assist bacteri-
ology staff at veterinary diagnostic laboratories to maintain their skills. On-going quality
control of all aspects of the diagnosis of bovine tuberculosis has been a feature of the
Australian bovine tuberculosis eradication program.
 L.A. Corner~Veterinary Microbiology 40 (1994) 53-63 63

Acknowledgements

The excellent technical skill of Ms. K. Lund and Ms. J. Krywult is gratefully acknowl-
edged. This work was supported by the Ausa'alian National Brucellosis and Tuberculosis
Eradication Campaign.

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