TMP E7

International journal of advanced scientific and technical research Issue 4 volume 1, January-February 2014
Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954
Prevalence of Bovine tuberculosis in Kassala state, Eastern the Sudan
Ayman,E.A 1.,. D.A.Salih 2 , M.M.Gumaa 3 ,M.M.Omer 3, Fatima Abbas 4, and

A. M. Ahmed 2
1 Kassala Veterinary Hospital-Kassala/Sudan

2Veterinary Research institute-Khartoum /Sudan PO Box 8067 ALAmarat
3 Regional veterinary Research-Kassala/Sudan
4 Faculty of Medicine, Tuberculosis centre, Kassala/Sudan
Abstract
This study was conducted to determine the prevalence of bovine tuberculosis (BT).The
number of tested cows 569 animals (140 local breeds and 429 cross breed). They were
randomly selected from 6 localities in Kassala State, Sudan, using comparative cervical
tuberculin test (CCTT), Rapid test, Indirect ELISA and PCR. A prevalence of 1.93% was
recorded in the study area using the CCTT.
The results of the different tests which were implemented in 6 locations showed that the
highest infection was in the Kassala 60% using PCR, while the lowest infection was in
Fato, Halfa Sugar farm and Khashelgirba 0% using CCTT.
Many epidemiological risk factors including housing conditions breed, and management
systems were assessed for their contribution to the prevalence of the disease.
Introduction
In most African countries, consumption of unpasteurized milk is a regular practice
(Ayele et al. 2004), leading to considerable risk of zoonotic infection with
Mycobacterium bovis and other mycobacteria. M. bovis has been isolated earlier
from unpasteurized milk and lesions of slaughtered cattle in Nigeria, as well as from
patients with pulmonary and extrapulmonary tuberculosis (Cadmus et al. 2006).
The epidemiology and public health significance of bovine TB in Africa remain
largely unknown; few laboratories are capable of differentiating M. bovis from M.
tuberculosis and other members of the M. tuberculosis complex (MTC).
None of the national reports submitted to the OIE and WHO by African member
states mention the importance of M. bovis in human TB cases. (Ayele et al, 2004).
R S. Publication, rspublicationhouse@gmail.com Page 238

Collins and Grange (1983) reported a lack of interest in typing human tubercle
bacilli in the laboratories of several countries, leading to underestimations of the
incidence of M. bovis. The primary sources of infection for humans are consumption
of unpasteurized milk and close association between humans and animals (Behr.,
1999, Daborn et al., 1996). The link between drinking milk from diseased cows and
the development of scrofula, cervical lymph node tuberculosis, was established in
mid-19th century when more than half of all cervical lymphadenitis cases in children
were caused by M. bovis.
Infection acquired through ingesting M. bovis is more likely to result in
non-pulmonary forms of disease.
Zoonotic tuberculosis is an important public health concern worldwide, especially
in developing countries, because of deficiencies in preventive and/or control
measures (Etter et al .,2006).
Isolation of the bacteria is the most reliable method for diagnosis of mycobacterial
disease (Nolte and Metchok, 1995), and culture is still internationally considered the
gold standard for detection of mycobacteria; and is more sensitive and is presently
the yardstick for diagnosis, but the time required and frequent negative results in
paucibacillary specimens are important limitation (Katoch, 2003; Haldar et al.,
2007). (Ayele et al., 2004). The serodiagnosis of tuberculosis has long been the
subject of investigation, but we still lack a test with widespread clinical utility. Any
new test should have a specificity of >90% .The several commercial assays available
are rarely used as the sole means of diagnosis because of unsatisfactory sensitivity
and specificity (Chiang et al., 1997; Pottumarthy et al., 2000). An advantage of
ELISA is its simplicity, but sensitivity is limited mostly because of the late and
irregular development of the humoral immune response in cattle during the course of
the disease.
Specificity is also poor in cattle when complex antigens such as tuberculin or M.
bovis culture filtrates are used. However, a comparison of antibody levels to PPD-B
and PPD-A has been shown to be useful in increasing specificity in the ELISA
(Griffin et al., 1993). Moreover, in M.-bovis-infected animals, an anamestic rise has
been described, resulting in better ELISA results 2–8 weeks after a routine
tuberculin skin test (Lyashchenko et al., 2004).
The standard method for detection of bovine tuberculosis is the tuberculin test,
which involves the intradermal injection of bovine tuberculin purified protein
derivative (PPD) and the subsequent detection of swelling (delayed
hypersensitivity) at the site of injection 72 hours later.

As the sensitivity of the test is less than 100%, it is unlikely that eradication of
tuberculosis from a herd will be achieved with only a single tuberculin test. It should
be recognized that when used in chronically infected animals with severe pathology,
the tuberculin test may be unresponsive. (Oie, 2009).
The polymerase chain reaction is a powerful technique that has rapidly become one
of the most widely used techniques in molecular biology because it is quick,
inexpensive and simple. The technique amplifies specific DNA fragments from
minute quantities of source DNA material, even when that source DNA is of
relatively poor quality. (Erlich, 1989).
Materials and Methods
Study area
Kassala State , lies between Latitudes 14°15´ and 17°15´ N and Longitudes 34°30´
and 37°E´ in eastern Sudan; it borders Eritrea and Ethiopia (Fig. 1). The total animal
population in this state according to the Administration of Animal Resources (Anon,
2006) is 3800553, animal species is as follows: 631957 cattle, 1457643 sheep,
1122073 goats and 588880 camels.
Tuberculin test
Bovine and avian purified protein drevatives, PPD (Synbiotics) were used as
injected antigens in this test.Each ml of the bovine and avian PPD contains 20000
IU.
The procedure was carried out as described in the OIE Terrestrial Manual (2009).
Briefly, the injection site on the mid-neck were clipped and cleaned. A
Rapid Test
One Step Bovine TB Antibody RAPID TEST was purchased from Labcare
Diagnostics Ltd. London UK.
A test result had seen as a purple band in the result window of the kit. Test result
interpreted within 20 minutes. Even if the intensity of the purple band were faint, it
had been interpreted as positive if it appears within 20 minutes. A color band
appeared in control line (c) to show that the test was working properly. Another
color band appeared in the result window this band was the Test line (T). The
presence of only one purple color band within the result window indicates a negative
result. The presence of two color bands (‘T” band and ‘C”) within the result window,
no matter which band appears first indicated a positive result.

Indirect ELISA
The procedure was carried out as described by Lilenbaum and Leila (2006)
We evaluated the alternative cut-off point by calculation of the mean + 2SDs
(Andrew et al., 2004).
Molecular Identification
Deoxyribonucleic (DNA) Extraction from milk samples
DNA was extracted from milk samples by Innu PREP DNA Mini Kit following the
protocol recommended by manufacturer’s (Analytikjena bio solutions, Germany).
Briefly, the cells were pelleted by centrifugation for 10 min at 5.000 × g (7.500 rpm),
then the supernatant was discarded and 400 µl of the lysis solution (TLS) and 25 µl
proteinase K were added to the pellet, mixed vigorously by pulsed vortexing for 5
second then incubated at 50 °C until the sample was completely lysed.
Subsequently, 400 µl of the binding solution (TBS) was added to the lysed sample,
mixed by brief vortexing for 15 second, then the samples were applied to the spin
filter located in a 2.0 ml receiver tube and centrifuged at 10.000 × g (12.000 rpm) for
2 minutes. Thereafter, the receiver tube was discarded and the spin filter was placed
into a new 2.0 ml receiver tube and 500 µl of washing solution (HS) was added, and
centrifuged at 10.000 × g (12.000 rpm) for 1 minute. Then the receiver tube was
discarded and the spin filter was placed into a new 2.0 ml receiver tube before 750 µl
of washing solution (MS) was added, and centrifuged at 10.000 × g (12.000 rpm) for
1 minute. Again, the receiver tube was discarded and the spin filter was placed into a
new 2.0 ml Receiver Tube. A step of centrifugation at maximum speed for 2 minutes
was carried out to remove all traces of ethanol, then the 2.0 ml receiver tube was
discarded and the spin filter was placed into a 1.5 ml elution tube. To elude the
DNA, 200 µl elution buffer (EB) was added to central of the spin filter, incubated at
room temperature for 1 minute, and then centrifuged at 6. 000 × g (8.000 rpm) for 1
minute. The DNA was stored at – 20 °C, until subjected to the subsequent
experiments.
Polymerase Chain Reaction (PCR)
The PCR reactions were performed in a total volume of 50 µl as follows: 25 µl

Green master mix, containing dNTPs, PCR buffer and Taq polymerase (Fermentas,
Germany), 16 µl H2O, 2 µl of each primer at a dilution of 10 pmol and 5 µl genomic
DNA. A multiplex PCR was carried out with the following primers common
forward CSB1 ('5-TTCCGAATCCCTTGTGA-3') and two reverse primers,
including M. bovis-specific, CSB2 ('5-GGAGAGCGCCGTTGTA-3') and M.
tuberculosis-specific, CSB3 ('5-AGTCGCGTGGCTTCTCTTTTA-3'). These

primers were designed by Bakshi et al. (2005). The thermo cycler program for was
as follows: 94 °C for 3 minute, then 35 cycles at 94 °C for 1 minute, 52.3 °C for 1
minute, and 72 °C for 1 minute, a final extension step 72 °C for 7 minute, and then
held at 4 °C. In each run, positive and negative controls were included. The positive
control was DNA extracted from M. tuberculosis culture from a human patient,
while the negative control was distilled water.
Detection of PCR product
The amplified fragments were separated by electrophoresis on 1.5% agarose gels. A

volume of 5 μl from each PCR reaction (containing the loading dye) was loaded on
the agarose gel. A volume of 5 μl of 100 bp DNA-ladder (Roth, Germany) was also
loaded. The gel was run at 70 V for 1 h and the separated fragments were viewed
using a UV-transilluminater and the results were documented.
Milk samples collection and processing and culture
About 30 ml of the last few streams of milk from the 4 quarters of CCTT positive
cows were collected into a sterile tube aseptically. The samples were kept in -20◦C
.The milk samples were processing by centrifuged at 3000 rpm for 15 min and the
supernatant discarded .The sediments were suspended in 2 ml of sterile
physiological saline solution (Ameni and Wudie., 2003), (Quinn et al ., 2002).
Smears for acid fast bacilli were made from each collected sample, after fixing by
flame and then stained using Ziehl –Neelsen . The samples were then cultured onto
Loenstein Jensen (LJ) medium, incubated at 370C for 16 weeks.
Results
Out of 569 cattle subjected tuberculin test, 11 (1.93%) showed positive results.
A total of 8 cattle from farms, which represented 72.7% of the positive cattle, 1 from
backyard (9.1%) and 2 from open grazing (18.2%). (Fig 3).
The results showed that Tagalobab had the highest percentage of positive results
according to tested number whereas Kassala city and Elgash area had the lowest
Percentage and no positive results were reported in Girba, Fato and Halfa sugar farm
(Fig 4).
Rapid Test: Out of 256 cattle subjected to this test, 80 (31.2%) showed positive results
(Table 1).

Table 1: Distribution of Rapid test results according to management system, housing conditions and
breeds among cattle in Kassala State
Variables Rapid test
No. Positive %
examined
Management Farms 158 52 32.9

system
Open 58 21 36.2
grazing
Backyard 40 7 17.5
Grand 256 80 31.2

Total
Breed Indigenous 51 20 39.2
Cross 205 60 29.3
Grand 256 80 31.2

Total
Housing 3 0 0 0
Condition
(Farm)
˃ 1.5 9 8 88.9
≤ 1.5 13 12 92.3
Grand 22 20 90.9
Total
Out of these positive results, 52 cattle (32.9%) were originated from farms, whereas
21 cattle (36.2%) were from open grazing, and 7 cattle (17.5%) were from backyard
(Fig 6).
Indirect ELISA
Out of 346 cattle subjected to this test, 9 (2.6%) showed positive results (Table 2).
Data from this test shown that 2(4.4%) were indigenous breed, 7(2.3%) were cross
breed. The results showed that 7 out of 9 positive results of this test were from
Kassala city, 1 from Girba and 1 from Elgash area .
Table 2: Distribution of indrect ELISA results according to management system, housing conditions
and breeds among cattle in Kassala State
Variables Indirect ELISA
No. Positive %
examined

system
Open 50 2 4
grazing
Backyard 31 1 3.2
Grand 346 9 2.6

Total
Breed Indigenous 45 2 4.4
Cross 301 7 2.3.
Grand 346 9 2.6

Total
Housing 3 0 0 0
Condition
(Farm)
˃ 1.5 11 1 9.1
≤ 1.5 13 5 38.5
Grand 24 6 25
Total
Molecular Identification
Results by PCR method showed that 12 (70.6%) of 17 milk samples were positive
(Table 3).
Out of 12 positive samples 11(64.7%) were positive for M.bovis, 7(41.2%) were
positive for M.tuberculosis whereas 6 (35.3%) were mixed infection.

Table 3: Distribution of PCR results according to management system, housing conditions and
breeds among cattle in Kassala State
Variables PCR
No. Positive %
examined

system
Open 2 2 100
grazing
Backyard 1 1 100
Grand 17 12 70.6
Total
Breed Indigenous 2 2 100
Cross 15 10 66.7
Grand 17 12 70.6
Total
Housing 3 0 0 0
Condition
(Farm)
˃ 1.5 5 3 60
≤ 1.5 9 6 66.7
Grand 14 9 64.3
Total
Table 4: Distribution of results according to locations
No Rapid
Location CCTT(A/B) ELISA(A/B) PCR(A/B)
collected Test(A/B)
Gash 122 3/122 14/36 1/69 2/2

Kassala 277 7/277 55/188 7/160 9/15
Fato 25 0/25 ND ND ND
Togolobab 10 1/10 1/7 0/5 1/1
Khashelgirba 26 0/26 10/25 1/17 ND
Halfa Sugar 109 0/109 ND 0/95 ND
Total 569 11/569 80/256 9/346 12/17
(A/B): Number positive/number examined
* ND: Not done

Table 5: Cross tabulation between CCTT and PCR results
PCR
Positive Negative Total

Positive 10 3 13
CCTT
Negative 1 3 4
Total 11 6 17
Growth culture:
No bacterial growth detected during the whole incubation period
Discussion
Out of 11 positive to CCTT 9 were negative to Indirect ELISA test, this may be
due to fact that antibodies tend to appear later than the cellular response.
On other hand 7 out of 9 positive to indirect ELISA were negative to CCTT, this
results can be justifying by saying that the cellular response is considered to be
strongest in the early stage of infection and decreases as the disease progresses. On the
other hand, antibodies tend to appear most frequently in advanced, long-dated or
disseminated disease, when there is a heavy antigenic load (Ritacco et al 1990). Also
antibody response, therefore, is useful to identify those animals that fail to respond to
the intradermal tuberculin test, known as anergic animals (Plackett et al 1989).
Since antibodies tend to appear later than the cellular response, the CCTT test should
be performed on all ELISA-negative animals, because they may be infected in the
earlier stages of the disease.
In our study, the agreement between CCTT and PCR was determined as good.
Milk samples obtained from animals that had been positive for CCTT was subject
to PCR assay in order to determine if such an assay would improve the efficiency
of diagnosis.
According to the tuberculin test results, the positivity rate for bovine tuberculosis
was identified as 1.9% (11/569) in the animals tested. Milk samples of 9 CCTT
positive cattle were analyzed with PCR, the results showed that 8 were positive to
PCR whereas one sample was negative.

In a study by Romero et al., (1999), of 184 positive to CCTT, 26 were positive to

PCR, he reported that PCR was more sensitive and specific test than the tuberculin
test.
Liebana et al., 1995, Gonzalez et al., 1999, Amadori et al., 2002, have
compared classical diagnostic methods with PCR in their studies and reported that
PCR was a more sensitive method. In our study, CCTT results were in close
correlation with the PCR analysis results. Therefore the 8 positive animals that had
tested on the CCTT and PCR, to some extent can be considered as cases of bovine
tuberculosis. The policy of test and slaughter is not yet adopted in the Sudan, so we
were not able examine the reactive animals at the postmortem level and hence we
did not take samples from their internal organs so we relied only on milk samples
of positive reactors.
Culture on LJ medium is still the gold standard of BTB diagnosis,(Roberts et
al.,1991),but in this study culture on LJ medium failed to show any growth for
mycobacterium cells ,this negative result justifying the need for more effective
procedure for BTB diagnosis especially at milk producing cows , PCR assay can
provide such procedure particularly
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International journal of advanced scientific and technical research Issue 4 volume 1, January-February 2014

Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954

Prevalence of Bovine tuberculosis in Kassala state, Eastern the Sudan

Ayman,E.A 1.,. D.A.Salih 2 , M.M.Gumaa 3 ,M.M.Omer 3, Fatima Abbas 4, and

1 Kassala Veterinary Hospital-Kassala/Sudan

R S. Publication, rspublicationhouse@gmail.com Page 238

R S. Publication, rspublicationhouse@gmail.com Page 239

R S. Publication, rspublicationhouse@gmail.com Page 240

The PCR reactions were performed in a total volume of 50 µl as follows: 25 µl

R S. Publication, rspublicationhouse@gmail.com Page 241

The amplified fragments were separated by electrophoresis on 1.5% agarose gels. A

R S. Publication, rspublicationhouse@gmail.com Page 242

Variables Rapid test

Management Farms 158 52 32.9

Grand 256 80 31.2

Breed Indigenous 51 20 39.2

Cross 205 60 29.3

Grand 256 80 31.2

Variables Indirect ELISA

Management Farms 265 6 2.3

Grand 346 9 2.6

Breed Indigenous 45 2 4.4

Cross 301 7 2.3.

Grand 346 9 2.6

R S. Publication, rspublicationhouse@gmail.com Page 244

Management Farms 14 9 64.3

Breed Indigenous 2 2 100

Table 4: Distribution of results according to locations

Gash 122 3/122 14/36 1/69 2/2

R S. Publication, rspublicationhouse@gmail.com Page 245

Table 5: Cross tabulation between CCTT and PCR results

Positive Negative Total

No bacterial growth detected during the whole incubation period

R S. Publication, rspublicationhouse@gmail.com Page 246

In a study by Romero et al., (1999), of 184 positive to CCTT, 26 were positive to

R S. Publication, rspublicationhouse@gmail.com Page 248

R S. Publication, rspublicationhouse@gmail.com Page 249

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