This study aimed to detect Mycobacterium bovis (M. bovis) in milk and dairy products in Egypt using polymerase chain reaction (PCR) targeting the Mce4A gene. Three hundred random samples were collected from Assiut, Egypt including raw milk, yogurt, cheese and butter. Isolation methods found M. bovis in 4% of samples from tuberculosis-positive animals and 2% from negative animals. M. bovis and other mycobacteria were also detected in 1-2% of dairy products. PCR was performed on 6 M. bovis strains previously identified, which confirmed the presence of the Mce4A gene. The results suggest using the Mce4A gene as a target for
2. 15 Sci. Cong. 2012, Fac. Vet. Med., Assiut Univ., Egypt
2
Polymerase Chain Reaction (PCR) proved to be a rapid, accurate,
sensitive and specific method for detection of M. bovis in regard to
conventional, biochemical and serological methods. Three hundred
random samples were collected from different localities in Assiut city
including 150 raw milk samples and some dairy products as locally
manufactured yogurt, Kareish cheese and cooking butter (50 samples for
each). The incidence of Mycobacteria was 4, 2 and 0% using Lowen
stein Jensen (L-J) medium Pyruvated and 2, 0 and 0% using L-J medium
Glycerinated for tuberculin positive reactors, tuberculin negative
reactors dairy animals and marketable milk samples, respectively. In
locally manufactured yogurt, Kareish cheese and cooking butter samples
the incidence was 4, 2, 2% for pyruvated media and 0, 2, 2% for
glycerinated media, respectively. Identification of isolates indicated M.
bovis (4%) and Mycobacteria other than tuberculosis (MOTT) (2%)
from tuberculin positive reactors and (2%) from tuberculin negative
reactors animals. M. bovis and MOTT could be detected in 1 (2%) of
locally manufactured yogurt, Kareish cheese and cooking butter and
failed detection in marketable milk samples. PCR was done to 6 M.
bovis strains previously identified by biochemical tests and positive
results were obtained. To confirm these isolates we suggest using
specific primer (Mce4A) as a specific primer for locally isolated strains.
More efforts are needed to enhance and promote farms and sale points of
milk.
INTRODUCTION
Tuberculosis (TB) is still a major public health problem in many
parts of the world despite multiple efforts to combat it (Isdore et al.,
2010). Every minute (somewhere in the world) four people die from
tuberculosis (Khansobis et al., 2002).W H O (2009) estimated 13.7
million people had active TB disease, with 9.3 million new cases and 1.8
million deaths; the annual incidence rate varied from 363 per 100,000 in
Africa to 32 per 100,000 in the Americas. The high prevalence of
Bovine Tuberculosis among the human population in Egypt is due to the
way of living of more than 20 million Egyptians at rural areas, where
those human and farm animals live side by side in close contact and the
insufficient extension service in the rural areas (Siam, 1992).
The polymerase chain reaction (PCR) showed more promising for
Mycobacterial spp. detection in clinical samples. This technique seemed
to have sensitivity equal or greater than that of the culture method. Thus
3. 15 Sci. Cong. 2012, Fac. Vet. Med., Assiut Univ., Egypt
3
PCR increases the rate of detection and will be a useful tool for control
programs especially when considering the lower sensitivity of diagnosis
by bacteriological culture, the occupational risk, technical difficulties
with the protocol and the prolonged time of incubation (Bermudez et al.,
2010). Mammalian cell entry (mce) was able to confer the ability to
invade HeLa cells and macrophages to E. coli cells transformed with
plasmids containing it. They found that the mce gene sequence contains
two open reading frames (ORF). The first (ORF1) has been shown to
contain the element responsible for attachment and entry into
mammalian cells; the second was shown to confer the ability to survive
inside cells. Kumar et al. (2008) demonstrated that recombinant mce4A
protein facilitates the invasion of non-pathogenic strain of E. coli into
non-phagocytic HeLa cells. They also observe that mce4A gene has a
role comparable to mce1A in the survival of recombinant E. coli in
human macrophages. This study aimed to detect specific gene in Egypt
that help in rapid diagnosis of tuberculosis.
MATERIAL AND METHODS
1- Tuberculin skin test: was done according to Ovdiennkop et al.,
(1987)
2- Isolation and identification of M. bovis from milk and some milk
products samples:
A- Collection of samples: A total of 300 random samples of raw milk
from (tuberculin positive, negative reactors and marketable milk) and
some milk products (yoghurt, kareish cheese and cooking butter) were
collected from different localities in Assiut Governorate.
B- Preparation of samples according to Neill et al., (1988) and
A.P.H.A, (1992):-
C- Identification of isolates: using Ziehl-Neelsen stain for acid-fast
bacilli and conventional methods (rate of growth, colonial morphology,
and pigmentation and biochemical properties) according to Brasil
(1994).
D- PCR analysis: (Sambrook et al., 1989).
1- Extraction of genomic DNA from field isolates: One hundred mg
(wet weight) of the cell pellet of each 9 isolate was re-suspended in
0.5ml TE buffer, allowed for 2 cycles freezing and thawing. The cells
were then homogenized in glass homogenizer and then incubated 4
hours at 37oC with 5ul lysozymes (final concentration 100μg/ml).
Proteinase-K was added 25μl/0.5 ml (final concentration 100μg/ml) and
4. 15 Sci. Cong. 2012, Fac. Vet. Med., Assiut Univ., Egypt
4
incubated for further 3 hours at 56 oC with shaking. DNA was then
extracted using Trizol reagent.
The supernatant was completely discarded and the DNA pellet was
washed twice with 0.1ml sodium citrate in 10% ethanol. At each time,
the DNA pellet was kept in the washing solution for 30 minutes at RT
with periodical mixing and centrifuged at 4000 rpm/5 minutes at 4oC.
Following the 2 washes, the DNA was re-suspended in 2ml of 75%
ethanol, kept at RT for 20 minutes with periodical mixing and then
centrifuged. The DNA pellet was finally dried briefly for 5 minutes
under vacuum and re-dissolved in 50ul of 8 mM NaOH. The pH was
adjusted at 8. Two µl of RNAase were then added and incubated at 37oC
for 1 hour.
2- Purification of DNA: Purification of DNA was done according to
manufacturer instruction.
3- Measuring DNA concentration and purity: One µl of DNA was
diluted with 49 µl dist. water and the optical density (OD) was measured in
a quarter cuvette at 260 and 280 nm.
4- PCR amplification of mce4A gene: In a 0.2 ml PCR tubes the
following reaction mixture was added: DNA tamplet (100 ng/µl) 10 μl,
Taq polymerase 5u/μl. (1 µl), 10x enzyme buffer 5 μl, dNTPs 2 μl then
added Q-Solution 10 μl, Primer 1 (1 μl), Primer 2 (1 µl): ~ Primer 1
(Sense): 5´-CAC-CTT-CCT-CAT-CCC-CTC-3´, ~ Primer 2 (Antisense):
5´-GAT-GAG-GGA-TTG-GAA-CAA-C -3´ and Bidist. water to 50 µl.
The mixture was placed in the thermal cycler (T- gradient, Biometra Inc,
Germany), which was programmed as follow: Initial denaturing applied at
(95oC/3 minute), number of Cycles (40 cycles), denaturing at (95oC/1
minute), annealing at (56 oC/45 seconds), extension at (72oC/1 minute) and
final extension at (72oC/10 minutes).
5- Agarose gel electrophoresis: Electrophoresis was done at 80 v/15
minutes and examined using UV Trans-illuminator.
RESULT
5. 15 Sci. Cong. 2012, Fac. Vet. Med., Assiut Univ., Egypt
5
Table 1: Incidence of Acid Fast Bacilli in milk and some milk products
samples examined microscopically using Ziehl-Neelsen stain
Type of
samples
Number of
examined
samples
Positive samples Negative
samples
No. % No. %
Milkfrom
tuberculin
positive
reactors
50 7 14 43 86
tuberculin
negative
reactors
50 3 6 47 94
Marketable milk 50 1 2 49 98
locally manufactured
yoghurt
50 4 8 46 92
Kareish cheese50 5 10 45 90
Cooking butter 50 2 4 48 96
Table 2: Incidence of Mycobacteria isolated from milk and some milk
products samples using conventional culture method.
Type of
samples
Number
of examined
samples
Lowenstein- Jensen mediaNo. of
+ve
samples
pyruvated glycerinated
+ve % +ve %
Milkfrom
tuberculin
positive
reactors
50 2 4 1 2 3
tuberculin
negative
reactors
50 1 2 0 0 1
Marketable milk 50 0 0 0 0 0
locally
manufactured
yoghurt
50 2 4 0 0 2
Kareish cheese50 1 21 2 1
Cooking butter 50 1 2 1 2 1
Table 3: Identification of Mycobacteria isolated from milk and some
milk products samples.
6. 15 Sci. Cong. 2012, Fac. Vet. Med., Assiut Univ., Egypt
6
Type of samples
No. of
samples
Positive culture
for AFB
Types of Mycobacteria
M. bovis MOTT
No. % No. % No. %
Milkfrom
tuberculin
positive
reactors
50 3 6 2 4 1 2
tuberculin
negative
reactors
50 1 2 1 2 0 0
Marketable milk 50 0 0 0 0 0 0
locally manufactured
yoghurt
50 2 4 1 2 1 2
Kareish cheese 50 2 4 1 2 1 2
Cooking butter 50 2 4 1 2 1 2
Total 300 10 3.3 6 2 4 1.3
Photo 1:- 1% agarose gel staining by 0.5μg/ml ethidium bromides
showing:-
-ve referred to a negative control (unamplified PCR products), +ve
referred to positive control, M referred to 100 base pair ladder marker.
Lanes: 1-6 positive for M. bovis by using mce4A gene (700bp) and
Lanes: 7-10 Mycobacteria other Than M. bovis.
DISCUSSION
7. 15 Sci. Cong. 2012, Fac. Vet. Med., Assiut Univ., Egypt
7
The results of AFB in milk samples examined by direct smear
using ZN stain was shown in Table (1). It is clear that the total positive
samples were 7 (14%), 3 (6%) and 1 (2%) in milk from tuberculin
positive reactors, tuberculin negative reactors and marketable milk
samples, respectively. While, the results of AFB in some milk products
samples examined by direct smear in locally manufactured yoghurt was
4 (8%), Kareish cheese 5 (10%) and Cooking butter 2 (4%). Nearly the
same detected by Shaimaa Shalapy (2009). The reason for such results
may be due to obtaining some milk samples from infected farms in
Assiut. Although the direct microscopical examination is easy, rapid and
cheap, it will be positive only in the presence of large number of bacilli
and it can't differentiate between tubercle bacilli and saprophytic AFB.
El-Guindi et al. (1980) and Dunn and Hodgson (1982) proved that
milk samples with enough viable tubercle bacilli excreted can infect the
milk of 100 clean cow to be of infectious level and confirmed by
Bastawrous (1992).
Mycobacteria was detected by culturing in 3 (6%) and 1 (2%) milk
samples from tuberculin positive and negative reactors while, failed
detection in marketable milk (Table 2). Higher results where detected by
Vekemans et al. (1999) who detected Mycobacteria in 26% of retailed
milk samples collected in markets in Burkina Faso, Munreo et al. (2000)
9.3% in Canadian cattle and Ameni et al. (2003) (13.3%). Clarice
Fujimura Leite et al. (2003) isolated Mycobacteria from 15 of 22
(68.2%) caseous lesions from livestock and from 23 of 128 (18%) milk
samples. Hamid et al. (2003) recorded 28.07% from milk of tuberculin
positive buffaloes and from milk of 25% tuberculin positive cows.
Richardson (1970) who reported that atypical mycobacteria were
probably present in the environment and may get entrance to udder by
contamination during animal lay down or during non-proper milking
with contaminated equipment, these environmental atypical
mycobacteria may succeeded to induce ascending infection and even
lead to positive tuberculin results.
The results of Mycobacteria by culturing was 2 (4%), 1 (2%) and 1
(2%) for locally manufactured yoghurt, Kareish cheese and Cooking
butter, respectively (Table 2). Higher values were recorded by Munreo
et al. (2000) and Clarice Fujimura Leite et al. (2003), but nearly
similar to Cousins and Dawson (1999), Hamid et al. (2003), Shirima
et al. (2003) and Faye et al. (2005).
In this study the isolated Mycobacteria from collected milk
samples were 4, 2 and 0% of M. bovis and 2, 0 and 0% of MOTT for
tuberculin positive, negative reactors and marketable milk, respectively
8. 15 Sci. Cong. 2012, Fac. Vet. Med., Assiut Univ., Egypt
8
while the results of locally manufactured yoghurt, Kareish cheese and
cooking butter were 2% of both M. bovis and MOTT for each (Table 3).
While, M. tuberculosis could not be detected from all samples. L-J
medium with Glycerol was used for the cultivation and differentiation of
human and bovine types of the tubercle bacillus. Maureen (1981) stated
that the growth of M. bovis could be stimulated by the addition of
sodium pyruvate to the medium.
The mce proteins are a family of invasion-like proteins located at
the Mycobacterial cell surface (Chitale et al., 2001 and Ahmad et al.,
2005). The mce gene sequence contains two open reading frames. The
first (ORF1) has been shown to contain the element responsible for
attachment and entry into mammalian cells; the second was shown to
confer the ability to survive inside cells (Ereny Markos, 2011).
Photo (1) showed (-ve) a negative control (unamplified PCR
products); (+ve) positive control; (M) 100 base pair ladder marker; lanes
(1-6) revealed to all M. bovis at 700bp (100%) while the lanes (7-10)
indicated MOTT and they were isolated from milk of tuberculin positive
reactors, locally manufactured yoghurt, Kareish cheese and Cooking
butter. These results were consistent with (Nassar et al., 2007 and
Ereny Markos, 2011). Higher results obtained by Sechi et al., (2000)
(86.7%) and Cristina et al., (2005) (76.5%).
PCR is a sensitive and fast diagnostic tool that can be used to
detect the agent in clinical samples in 48 h, but the presence of inhibitors
in samples can interfere with its performance (Haddad et al., 2004;
Singh et al., 2004 and Brasil, 2005) although, the sensibility of PCR
from culture is more accurate (Sakamoto, 1997) and more confirmatory
for isolates. Genetic elements specific to virulence of the target species
would constitute attractive probe targets. The presence of virulence
factors can help to establish the disease potential of different samples
(Lang et al., 1994). The genetic elements responsible for the virulence
of Mycobacteria are not yet well established. Many virulence genes of
various levels of complexity are probably found in the MTBC. Also,
Stuart et al. (1993) recorded that the PCR of samples prepared by the
chaotrope-silica method had a sensitivity of 75% and a specificity of
100%, whereas, PCR of samples prepared by the chloroform method had
a sensitivity of 92% and a specificity of 100% when compared with the
sensitivities and specificities of the combined classical microbiological
methods for the diagnosis of tuberculosis. It is concluded that the PCR
was at least as sensitive as microscopy, but had greater specificity
because samples with atypical mycobacteria were not detected by PCR.
9. 15 Sci. Cong. 2012, Fac. Vet. Med., Assiut Univ., Egypt
9
REFERENCE
A.P.H.A. (American Public Health Association) (1992): Standard
Methods for the Examination of Dairy Products. 16th Ed.,
American Public Health Association, New York.
Ahmad, S.; El-Shazly, S.; Mustafa, A.S. and Al-Attiyah, R. (2005): The
six mammalian cell entry proteins (mce3a–F) encoded by the mce3
operon are expressed during in vitro growth of M. tuberculosis.
Scand. J. Immunol., 62: 16–24.
Ameni, G.; Bonnet, P. and Tibbo, M. (2003): Across sectional study of
bovine tuberculosis in selected dairy farms in Ethiopia. Inter. J.
App. Res. Vet. Med., 1 (4): 253-258.
Bastawrous, A.F. (1992): Occurrence and Significance of Typical
Mycobacteria in Milk and Some Dairy Products. Ph. D. Thesis,
Fac. Vet. Med., Zagazig Uni.
Bermudez, H.R.; Renteria, E.T.; Medina, B.G.; Hori-Oshima, S.; De
La Mora, V.A.V.; Lopez, V.G.; Yu, W.L.; Pio, G.R.; Herrera,
J.C.; Pujol, C. and Nielsen, K. (2010): Correlation between
histophatological, bacteriological and PCR diagnosis of bovine
tuberculosis. J. Animal Vet. Advances, 9 (15): 2082-2084.
Brasil (1994): Manual de Bacteriologia da Tuberculose, Centro de
Referência rofessor Hélio Fraga, Fundação Nacional de Saúde,
Ministério da Saúde, Rio de Janeiro, 114.
Brasil (2005): Ministério da Agricultura, Pecuária e Abastecimento.
Programa Nacional de Controle e Erradicação de Brucelose e
Tuberculose. www.agricultura. gov. br.
Chitale, S.; Ehrt, S.; Kawamura, I.; Fujimura, T.; Shimono, N.;
Anand, N.; Lu, S.; Cohen-Gould, L. and Riley, L.W. (2001):
Recombinant M. tuberculosis protein associated with mammalian
cell entry. Cell Microbiol., 3: 247- 254.
Clarice Fujimura Leite, Q.; Ivone Anno, S.; Sergio de Andrade Leite,
R.; Eliana, R.; Glenn, P.M. and Robert, C.C. (2003): Isolation
and identification of Mycobacteria from livestock specimens and
10. 15 Sci. Cong. 2012, Fac. Vet. Med., Assiut Univ., Egypt
10
milk obtained in Brazil. Mem. Inst. Oswaldo Cruz. Rio de
Janeiro., 98 (3): 2003.
Cousins, D.V. and Dawson, D.J. (1999): Tuberculosis due to M. bovis
in the Austerlian population: cases recorded during 1970-1994. Int.
J. Tuberc. Lung Dis., 3 (8): 715-721.
Cristina, P.D.; Clarice, Q.; Leite, E.; Karina, A.D.; Klaudia, D.;
Gonçalves, J.; Ana, L.A. and Rosa, O. (2005): M. bovis
identification by a molecular method from post-mortem inspected
cattle obtained in abattoirs of Mato Grosso do Sul, Brazil. Mem.
Inst. Oswaldo. Cruz, 100 (7): 749-752.
Dunn, B.L. and Hodgson, D.J. (1982): Atypical mycobacteria in milk. J.
App. Bacteriol., 52: 373-376.
El-Guindi, S.M.; Ahmed, O.L.; Awad, W.M. and El-Saban, M.S.
(1980): Incidence of bovine and human tubercle bacilli in milk and
milk products. Agr. Res. Rev., 58 (7): 75-84.
Faye, B.; Castel, V.; Lesnoff, M.; Rutabinda, D. and Dhalwa, J. (2005):
Tuberculosis and brucellosis prevalence survey on dairy cattle in
Mbarara milk basin (Uganda). Preventive Vet. Med., 67 (4): 267-
281.
GOVS (1992): General Organization for the Veterinary Services.
Ministry of Agri, Egypt.
Haddad, N.; Mansseloto, M. and Durand, D. (2004): Molecular
differentiation of M. bovis isolates. Res. Vet. Sci., 1-18.
Hamid, J.; Puran, D. and Asif, S. (2003): Bovine tuberculosis in dairy
animals at Lahore, threat to the public health. http://priory.com/
vet/ bovinetb.htm
Isdore, C.S.; Jongseok, L.; Caroline, A.´G.; Eun-Jin, C.; Ji-im, L.;
Vignesh, R.; Eun, G.L.; Jin, H.M.; Matthew, W.C.; Lisa, C.G.;
Jin, H.K.; Hyung, S.K.; Soohee, H.; Seok-Yong, E.; Seung, K.P.;
Hyeyoung, L.; Philip, S.; Sang-Nae, C.; Laura, E.V. and Clifton,
E.B. (2010): Genetic diversity of M. tuberculosis isolates from a
tertiary care tuberculosis hospital in south Korea. J. Clin.
Microbiol., 2 (48): 387–394.
11. 15 Sci. Cong. 2012, Fac. Vet. Med., Assiut Univ., Egypt
11
Khansobis, S.; Escuyer, V.E. and Chatterjee, D. (2002): Emerging
therapeutic targets in tuberculosis: post genomic era. Exprt. Opin.
Ther. Targets. 6 (1): 21.
Kumar, V.; Abbas, A.K.; Fausto, N. and Mitchell, R.N. (2008):
Robbins Basic Pathology (8th Ed.). Saunders Elsevier. Pp. 516–522.
Lang, L.A.; Tsai, Y.L.; Mayer, C.L.; Patton, K.C. and Palmer, C.J.
(1994): Multiplex PCR for the detection of the heat-labile toxin
gene and shiga-like toxin I and II genes in E. coli isolated from
natural waters. App. Environ. Microbiol., 60: 3145–3149.
Maureen, C.V. (1981): Mycobacteria. Printed in Great Britian by John
Wright and sons Ltd, at the stone bridge press. Bristol B545.
Munreo, F.A.; Dohoo, I.R. and Ncnab, W.B. (2000): Estimates of
within herd incidence rates of M. bovis in Canadian cattle and
cervids between 1985 and 1994. Preventive Vet. Med., 45 (3-4):
247-56.
Nassar, A.F.C.; Miyashiro, S.; Oliveira, C.G.; Pacheco, W.A.; and
Ogata, R.A. (2007): Isolation and identification of bovine
tuberculosis in a Brazilian herd (São Paulo). Mem. Inst. Oswaldo
Cruz., 102 (5):112 – 120.
Neill, S.D.; Brien, J.J. and MacCracken, R.M. (1988): M. bovis in the
anterior respiratory tract in the herds of tuberculin reacting cattle.
Vet. Rec., 122: 184-186.
Ovdiennkop, N.P.; Shchur evskii, V.E.; Naimanor, A.K.; Yokuskeva,
O.V.; Sharov, A. and Planikov, E.S. (1987): Frequency of
tuberculin injection in cattle. Vet - Muscow, us sr., 8: 29-33.
Richardson, A. (1970): Bovine mastitis associated with M. Smegmatis
and untypable mycobacterium. Vet. Rec., 86: 497-498.
Sambrook, J.; Fritscgh, E.F. and Meniates, T. (1989): Molecular
Cloning. A Laboratory Manual. 2nd Ed. ColdSpring, Harbor,
141.Siam, M.A. (1992): Public Health Importance of Bovine
Tuberculosis. Proc. Of Int. Conf. On Animal Tuberculosis, GOVS,
Cairo, Egypt, Pp. 195-197.
12. 15 Sci. Cong. 2012, Fac. Vet. Med., Assiut Univ., Egypt
12
Sechi, L.A.; Dupre, I.; Leori, G.; Fadda, G. and Zanetti, S. (2000):
Distribution of a specific 500-base-pair fragment in M. bovis
isolates from Sardinian cattle. J. Clin. Microbiol., 38: 3837-3839.
Shaimaa Shalapy, M.I. (2009): Are Milk and Milk Products Still
Important Vehicle for Transmitting Tubercle Bacilli to Human? M.
V. Sc. Thesis, Fac. Vet. Med. Kafr El-sheikh Uni.
Shirima G.M.; Kazwala R.R. and Kambarage D.M. (2003): Prevalence
of bovine tuberculosis in cattle in different farming systems in the
eastern zone of Tanzania. Preventive Vet. Med., 57 (3): 167-72.
Singh, S.K.; Verma, R. and Shah, D.H. (2004): Molecular finger
printing of clinical isolates of M. bovis and M. tuberculosis from
India by Restriction Fragment Length Polymorphism (RFLP). J.
Vet. Sci., 5: 331-335.
Stuart, M.W.; Ruth, M.; Pamela, M.N.; Peter, D.; Godfrey, F. and
Alister, V. (1993): Progress toward a simplified PCR and its
application to diagnosis of tuberculosis. J. Clin. Microbiol., 31 (4):
776-782.
Vekemans, M.; Cartoux, M. and Diagbouga, S. (1999): Potential
source of human exposure to M. bovis in Burkina Faso, in the
context of the HIV epidemic. Clin. Microbiol. Infect., 5: 617-612.
WHO (World Health Organization) (2009): "The Stop TB Strategy,
case reports, treatment outcomes and estimates of TB burden".
Global tuberculosis control: epidemiology, strategy, finances. Pp.
187–300.
