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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ABSTRACT: The World Health Organization (WHO) estimates that human tuberculosis (TB) incidence and deaths for 1990 to 1999 will be 88 million and 30 million, respectively, with most cases in developing countries. Zoonotic TB (caused by Mycobacterium bovis) is present in animals in most developing countries where surveillance and control activities are often inadequate or unavailable; therefore, many epidemiologic and public health aspects of infection remain largely unknown. We review available information on zoonotic TB in developing countries, analyze risk factors that may play a role in the disease, review recent WHO activities, and recommend actions to assess the magnitude of the problem and control the disease in humans and animals.Emerging infectious diseases 4(1):59-70. · 6.17 Impact Factor
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ABSTRACT: In order to gain a better understanding of the molecular epidemiology of Mycobacterium bovis isolates in Cameroon, 75 isolates of M. bovis collected in three provinces of northern Cameroon were studied by spoligotyping. For 65 of these isolates, typing was also carried out by pulsed-field gel electrophoresis (PFGE) with DraI, and 18 of the isolates were also typed by restriction fragment length polymorphism (RFLP) analysis with probe IS6110-RHS. Molecular typing of the isolates by these techniques revealed a high degree of homogeneity, with 10 spoligotypes for 75 isolates, four PFGE profiles for 65 isolates, and three RFLP types for 18 isolates. Some types were present in the three different provinces, while some were confined to one or two areas. These results suggest that geographical mapping of M. bovis strains could be helpful for the control of bovine tuberculosis at the regional level. An interesting feature of all the spoligotypes was the absence of spacer 30, suggesting a common origin for all of the Cameroon isolates tested; an evolutionary scenario for the isolates is discussed. In addition, a comparison of the three techniques showed that for M. bovis strain differentiation in Cameroon and in surrounding countries, spoligotyping would be a more discriminating and practical tool for molecular typing than the other two techniques used in this study.Journal of Clinical Microbiology 02/2001; 39(1):222-7. · 4.15 Impact Factor
Article: Rapid and specific detection of the Mycobacterium tuberculosis complex using fluorogenic probes andreal-time PCR.[show abstract] [hide abstract]
ABSTRACT: A rapid and sensitive strategy for the specific identification of Mycobacterium tuberculosis (TB) was designed and evaluated using crude mycobacterial lysates. The speed of real-time polymerase chain reaction (PCR) was combined with the sensitivity of fluorogenic probes to confirm the presence of mycobacteria as well as specifically identify the presence of members of the mycobacteria tuberculosis complex (MTC) in a single-tube assay. Oligonucleotides were designed to amplify the internal transcribed spacer (ITS) from several mycobacterial species. Specific fluorogenic probes were included in the PCR reaction for the identification of TB as well as Mycobacterium bovia and Mycobacterium africanum in bacterial lysates. The combination of TB-specific fluorogenic probes with real-time PCR formed an approach determined to be fast (less than 40 min), sensitive (less than 800 copies of DNA) and reliable for the specific detection of the MTC. Our data demonstrate the use of real-time PCR and fluorogenic probes in a rapid and sensitive assay to distinguish members of the MTC from other mycobacterial species.Molecular and Cellular Probes 01/2002; 15(6):375-83. · 2.08 Impact Factor
Myc obac terium
bovis Isolates from
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
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
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
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
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).
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
770Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 5, May 2006
Figure. Spoligotypes obtained from 55 Mycobacterium bovis isolates from Chadian zebus.
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.
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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Address for correspondence: Markus Hilty, Socinstrasse 57, PO Box,
Swiss Tropical Institute, 4002 Basel, Switzerland; email: Markus.Hilty@
M. bovis Isolates from Tuberculous Lesions
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 5, May 2006 771