Mycobacterium bovis isolates from tuberculous lesions in Chadian zebu carcasses.

Abstract

This slaughterhouse study in Chad shows higher proportions of Mycobacterium bovis isolates among Mbororo than Arabe zebu cattle. Spoligotyping shows a homogenetic population structure for M. bovis and lack of spacer 30, as were found in neighboring Cameroon and Nigeria. This finding suggests transborder and ongoing transmission between cattle.

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Myc obac terium
bovis Isolates from
Tuberc ulous
Lesions in Chadian
Zebu Carc asses
Colette Diguimbaye-Djaibé,*1Markus Hilty,†*1
Richard Ngandolo,* Hassane H. Mahamat,* Gaby
E. Pfyffer,‡ Franca Baggi,§
Glyn Hewinson,¶ Marcel Tanner,†
Jakob Zinsstag,† and Esther Schelling†
This slaughterhouse study in Chad shows higher pro-
portions of Mycobacterium bovis isolates among Mbororo
than Arabe zebu cattle. Spoligotyping shows a homogenet-
ic population structure for M. bovis and lack of spacer 30,
as were found in neighboring Cameroon and Nigeria. This
finding suggests transborder and ongoing transmission
between cattle.
I
east (Ouaddaï region) (1) and 16.9% (95% CI
10.4%–23.5%) in the west (Chari-Baguirmi and Kanem
regions) (2). The latter comparative intradermal tuberculin
study was conducted with 34 additional transhumant
herds; a prevalence of 11.5% (CI 6.9%–18.5%) was found
when herds were considered as random effect in the model.
More tuberculin reactors were found among Mbororo than
Arab zebus (p = 0.02). In the slaughterhouse of Farcha in
N’Djaména, 90% of slaughtered cattle are of the Arab zebu
breed, 7% Mbororo zebu, and 3% Kouri (3). Previous
slaughterhouse studies showed that bovine tuberculosis
(TB) is an important cause of condemnation (i.e., if a car-
cass is fully condemned, the whole carcass is destroyed
[≈9% of all inspected cattle carcasses]) (4). Aretrospective
study on causes of condemnation after meat inspection
showed that most carcasses with tuberculous lesions were
detected from July to November and that more Mbororo
cattle than other breeds had TB-like lesions (42/60 vs.
132/1,539) (5). The diagnosis of suspected bovine TB was
based on sighting of typical macroscopic lesions of the
organs during meat inspection.
n Chad, prevalences of tuberculin-positive cattle are
0.8% (95% confidence interval [CI] 0.2%–1.4%) in the
In Chad, until this study was undertaken, bovine TB
was not confirmed by isolation or molecular characteriza-
tion of the causative agent, Mycobacterium bovis. This
organism is recognized as a zoonotic pathogen that infects
many persons, particularly in the developing world. The
highest prevalence of coinfection with bovine TB and
HIV/AIDS is also in the developing world (6). Our study
was aimed at isolating the first M. bovis isolates from spec-
imens of Mbororo and Arab cattle in the newly setup
mycobacteriology unit of the veterinary laboratory of
Fracha, at characterizing the isolates with molecular meth-
ods, and at comparing the isolates with those from
Cameroon (7).
The Study
From July 1 to August 31, 2002, a total of 727 of 10,000
cattle carcasses at the slaughterhouse of Farcha were con-
demned because of TB-like lesions on meat inspection.
The overall prevalence of suspect lesions was 7.3%. Asig-
nificantly higher (p = 0.04) proportion of lesions was
found among Mbororo (8.2%; 212/2,596) than Arab (7%;
515/7,397) cattle (8). Lesions were mainly found in the
lymph nodes and lungs (Table).
Specimens from 201 affected organs (lymph nodes,
lungs, and liver) of 199 randomly selected carcasses were
collected for further processing along with the following
information: breed, sex, partial or total condemnation of
the carcass, date of collection, and nature of specimen (8).
The geographic origins of the cattle could not be evaluat-
ed as they were brought to the slaughterhouse by traders
from local livestock markets. In the subsample of 199 ani-
mals, entire condemnation of the carcass in comparison to
partial condemnation occurred more often among Mbororo
than Arab cattle (19/75 vs. 11/124, χ2, p = 0.002). A high-
er proportion of Mbororo cattle with bovine TB infection
was also observed in Cameroon (9); this finding may indi-
cate that Mbororo are more susceptible to M. bovis strains
in the 2 Central African countries.
The 201 collected specimens were washed 3 times with
sterile, distilled water. Tissue samples were cut into 5 or 6
pieces and put in a sterile plastic bag containing 10 mL
sterile saline for homogenization. Samples were homoge-
nized in a blender for 1 min; this process was repeated 3
times. Ten milliliters of the suspension was decontaminat-
ed with N-acetyl-L-cysteine sodium hydroxide (0.5%
NALC–2% NaOH) (10), and 0.25mL was injected onto 2
Lowenstein-Jensen slants, l containing glycerol (0.75%)
and l containing pyruvate (0.6%). In addition,
Middlebrook 7H9 medium containing oleic acid-albumin-
dextrose-catalase and PANTA (polymyxin, amphotericin
B, nalidixic acid, trimethoprim, azlocillin) were injected
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 5, May 2006769
1These authors contributed equally to this study.
*Laboratoire de Recherches Vétérinaires et Zootechniques de
Farcha, N’Djaména, Chad; †Swiss Tropical Institute, Basel,
Switzerland; ‡Kantonsspital Luzern, Luzern, Switzerland;
§National Centre for Mycobacteria, Zurich, Switzerland; and
¶Veterinary Laboratories Agency, Weybridge, United Kingdom

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with 0.5mL of the decontaminated suspension. Injected
media were incubated at 37°C (without CO2) for 8 weeks.
Growth of mycobacteria was confirmed by smear (stained
by the Ziehl-Neelsen method) and acid-fast–positive
colonies were subcultured. Three biochemical tests (11)
were used to distinguish between M. tuberculosis complex
and nontuberculous mycobacteria. Results were confirmed
by real-time polymerase chain reaction (10).
Overall, M. bovis was isolated from more than one
fourth of tissue samples and in 42% of all positive cultures.
Significantly more M. bovis isolates were obtained from
Mbororo zebu (30/75) than from Arab zebu (26/124) (p =
0.004). The difference remained significant when the type
of condemnation and type of organ were included in a mul-
tivariate logistic regression model.
Spoligotyping, as described (12), was used as a tool for
identifying M. bovis within the M. tuberculosis complex
(lack of spacers 3, 9, 16, and 39–43) but also yielded
insights into the epidemiology of M. bovis. In total, 12 dif-
ferent spoligotypes were found among the 55 M. bovis iso-
lates; 51 (92.7%) of 55 isolates were in 8 clusters (>2
strains), which showed a homogenous population structure
(Figure).
The predominant spoligotype in our study was SP1,
with a cluster of 22 strains (40%), as was the case in the
study of Cameroon (7). SP1 that lacks spacer 30 corre-
sponds to C1; 2 other clusters described in Cameroon (C1
and C5) were also found in Chad (SP2 and SP4). The find-
ing of a high proportion of the same spoligotypes in the 2
countries indicates cross-border movement of cattle. Asub-
stantial degree of recent transmission of M. bovis strains
among cattle is supported by the apparently high preva-
lence (7%) of TB-like lesions at the slaughterhouse in
N’Djaména. However, the homogeneity of bovine strains
could also be due to the absence of introduction of new
spoligotypes in this particular area. Certain Cameroonian
clusters (C7, C8, C9, and C10) (7) were only detected in the
Adamaoua region, not in northern Cameroon or our
Chadian study. The established measures of the
Cameroonian government to prevent movement of cattle
between the Adamaoua and the 2 northern regions appear
effective. As to other neighboring countries, a recent publi-
cation describes 15 M. bovis isolates from cattle in Nigeria,
and these also lack spacer 30 (13). This feature seems to be
a characteristic of M. bovis strains in Central Africa.
Fifteen strains (8 from Arab and 7 from Mbororo zebu)
were typed with the IS6110 restriction fragment length
polymorphism (14) method, of which 11 and 4 isolates
contained 2 or 1 band, respectively (data not shown).
Therefore, Chadian M. bovis strains belong to low IS6110
copy number strains. Strains lacking spacer 30 had a band
at 1.9 kb, in accordance with the findings in Cameroon (7).
No association was found between the number of bands
and the cattle breed. IS6110 typing indicated 6 clusters
and, thus, was of lower discriminatory power than spolig-
otyping. In a recent study, variable number of tandem
repeat typing was more discriminatory for Chadian M.
bovis strains than IS6110 and spoligotyping (15).
Conclusions
The first mycobacterial laboratory established in Chad
confirmed bovine TB in Chadian herds by culturing and
characterizing M. bovis. A high ongoing and cross-border
transmission of M. bovis in cattle is suspected, but further
TUBERCULOSIS DISPATCHES
770Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 5, May 2006
Figure. Spoligotypes obtained from 55 Mycobacterium bovis isolates from Chadian zebus.

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molecular epidemiology studies are needed to analyze its
modes and risk factors. The apparently higher susceptibil-
ity of Mbororo zebus to M. bovis infection should be fol-
lowed-up with immunologic assays.
Acknowledgments
We thank the technicians of the National Center for
Mycobacteria, the Swiss Tropical Institute, and the “Laboratoire
de Recherches Vétérinaires et Zootechniques de Farcha” who
contributed to the project. We thank Véronique Vincent for com-
plementary analyses and Steve Gordon for advice and discussion.
The Swiss National Science Foundation is acknowledged
for financial support. This work received support from the
National Centre of Competence in Research North-South IP-4.
Dr Diguimbaye is head of the human and animal TB unit at
the Laboratoire de Recherches Vétérinaires et Zootechniques de
Farcha in Chad. One of her research interests is the evaluation of
new TB diagnostics.
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unibas.ch
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