Bovine tuberculosis (bTB) is a zoonotic disease with public health significance. The maximum detection of bovine TB in cattle populations in Egypt is vital to understand its epidemiology and zoonotic potentials and to achieve significant reduction and control of the disease in livestock. The tuberculin skin tests (TST) are currently the best available techniques for international field diagnosis of bTB in live animals [2, 21] and it is based on delayed hypersensitivity reactions. The results of tuberculin skin testing are presented in In Table (1) where out of 3750 tuberculin-tested cross-bred dairy cattle, 69 were found to be positive reactors with a prevalence rate of 1.8%.
Farm
|
Total no. of examined animals
|
Positive tuberculin
|
Number
|
%
|
1
|
170
|
2
|
1.2
|
2
|
220
|
5
|
2.3
|
3
|
290
|
7
|
2.4
|
4
|
140
|
3
|
2.1
|
5
|
320
|
8
|
2.5
|
6
|
500
|
12
|
2.4
|
7
|
410
|
11
|
2.7
|
8
|
250
|
3
|
1.2
|
9
|
180
|
2
|
1.1
|
10
|
290
|
4
|
1.4
|
11
|
370
|
5
|
1.4
|
12
|
155
|
2
|
1.3
|
13
|
225
|
2
|
0.9
|
14
|
110
|
2
|
1.8
|
15
|
120
|
1
|
0.83
|
Total
|
3750
|
69
|
1.8
|
Table (1): Tuberculin skin test result in dairy farm cattle from some Egyptian Governorates.
This is comparatively lower than that recorded by other investigators in Egypt [22], (6.9%), [23], (4.6%), and [24], (2.2%) and in some other African countries as in Ethiopia (11.6%) [25], and in Chad (8%) [26]. This noticable reduced incidence might be attributed to the fact that the farms included in this research perform the tuberculin test regularly and applied test and slaughter strategy [27]. On the other hand, the prevalence rate recorded in the present study is comparatively higher than that reported by other investigators in other countries, 0.9% to 1.3% in Tanzania [28, 29], respectively, 0.54 % in Venezuela [30], 0.4 %, in New Zealand [31] 0.05 to 0.15 % in Australia [32] and 0.02 % in Japan [33]. The postmortem findings of the slaughtered TB reactors revealed that 73.9% of these animals showed visible lesions. At the same time the percentage of tuberculin reactors with non-visible lesions were 26.1%, Table (2).
Total No. of animals
|
Visible lesions
|
Non- visible lésions
|
Generalized
|
Head
|
Pulmonary
|
Digestive
|
Mixed
|
69
|
No.
|
%
|
No.
|
%
|
No.
|
%
|
No.
|
%
|
No.
|
%
|
No.
|
%
|
8
|
11.6
|
5
|
7.2
|
22
|
31.9
|
6
|
8.7
|
10
|
14.5
|
18
|
26.1
|
Table (2): Result of Postmortem examination of tuberculin positive slaughtered cattle.
These results agreed with those reported by [34, 35]. On the other hand, a much higher percentage was recorded by [36] who reported (86.9%) in Queensland, [37]. In Egypt Lower percentage was also reported by [38], (46%), [39], (52.7%), and [40], (40.8%). The percentage of tuberculin positive animals with non-visible lesions was attributed to sensitization by atypical mycobacteria or even closely related microorganisms especially the members of the genus Nocardia or a combination of Liver Fluke infestation with saprophytic mycobacteria [41, 42]. Moreover, [43, 44] attributed the cause of non-specific reaction to the assumption that, those animals might be slaughtered at early stage of the disease where the tuberculous lesions were still invisible, or lesions may be found in parts of the body not routinely examined in the carcass such as bone or brain. The relation between the postmortem findings in different ages of tuberculin reactor cattle is presented in Table (3).
Table (3): Results of correlation between age of animal and PM finding of the slaughtered cases
The percentage of reaction-positive animals increased with age, reaching a maximum in animals over 60 months old. Similar studies in Great Britain recorded an increase of incidence of bTB with increased age [45]. It was suggested earlier by [46] that, the high positivity of the old age might be due to the reduced number γ and δ T cells responsible of the anti-mycobacterial immunity. [These cells are predominantly found in the circulation of young calves. It has also been suggested that increased incidence of TB in older animals can be due to a waning of protective capability in aged animals [47, 48]. The correlation between site of lesion and the isolated mycobacteria species is shown in Table (4).
No. of processed tissue samples
|
Bacteriological isolation (52 isolates)
|
M. bovis
|
Unidentified mycobacteria
|
No.
|
%
|
Slow growers
|
Rapid growers
|
No.
|
%
|
No.
|
%
|
69
|
48
|
69.6
|
3
|
4.3
|
1
|
1.4
|
Table (4): Result of bacteriological isolation and identification of mycobacteria isolated from tuberculin positive cattle.
The total acid-fast bacilli isolated from the 69 slaughtered cattle were 52 isolates (75.4%). According to the morphological characters, growth rate, pigmentation, growth at different temperatures and biochemical tests these isolates were identified into 48 (69.6%) M. bovis, 1 unidentified rapid grower strain (1.44%) and 3 (4.3%) unidentified pigmented slow grower strains. The recovery rate of M. bovis figured up to 69.6% but other authors reported a lower M. bovis recovery rates, 14.8% by [49], 20.2% by [50], 41% by [51], 35.4% by [52], 29.1% by [53], 5.6% by [54], and 42.9% by [55]. On the contrary, [56], in Korea reported a much higher isolation rate (92.1%), in the republic of Korea. These results depend mainly on the actual disease status present in the tested herd and to some extent on the experience of the investigators as well as the technique used for decontamination of tissue specimens. Data presented in Figure (2), shows the relation between the post-slaughter findings of positive tuberculin reactors and the type of mycobacteria recovered from the 69 carcasses, M. bovis isolates, rapid growers isolates and slow growers pigmented isolates were recovered at rates of 69.6 %, 4.3%, and 1.44 %, respectively. M. bovis was isolated at a rate of 100% from generalized TB, 91% from pulmonary TB, 81.8% from Extra Pulmonary TB, 90% from mixed TB, and 11.1% from non-visible lesion (NVL) reactors. These results coincide with those reported by [57] who isolated M. bovis from NVL reactors. On the other hand, the recovery rate of typical mycobacteria rapid growers and slow growers pigmented was 16.7% of the extra-pulmonary for both of them and 33.3% of the mixed for the rapid growers. None of the cases of generalized tuberculosis yielded atypical mycobacteria, since M. bovis has a tendency to induce progressive lesions and generalization [44, 58]. All cases with generalized TB yielded M. bovis, a finding indicating the high susceptibility of cattle to M. bovis infection. Comparison between bacteriological examination and serological testing by lateral flow kits of tuberculin-positive cattle are shown in Figure (3).
The results of the application of lateral flow kits on sera from tuberculin-positive cattle:
The One- step Anigen Rapid Bovine Antibody Test (IQRT) employed recombinant M. bovis MPB70 antigen specific for M. bovis as capture and detector antigen. It is important to also mention that serological testing was conducted prior to TST because the latter can boost antibody responses in infected cattle and emphasizes the importance of timing of collection of blood samples on the interpretation the test [59]. This Anigen TB antibody test kit has detected 43.8% of tuberculin positive cattle against 69.6% that were confirmed by bacterial isolation of M. bovis. While the other lateral flow assay Ubio quick VET test is a single-directional lateral-flow serological test, which was produced to provide a quick determination of the presence of M. bovis antibody [60, 61] has detected zero % of tuberculin positive cattle. Comparison between the result of ELISA test, rapid lateral flow test and bacteriological isolation of tuberculin positive cattle showed that 8 tuberculin positive reactors with generalized tuberculous lesions were bacteriologically positive (M. bovis isolation) and ELSIA positive using bovine PPD as coating antigen (100%).
As shown in Figure (4), out of 22 tuberculin positive reactors 10 (45.5%) showed pulmonary lesion, out of 11 tuberculin positive reactors 3 (27.3%) showed extra-pulmonary lesions (head and digestive), and Out of 10 tuberculin reactors showing mixed TB lesions (pulmonary and extra pulmonary), 5 (50%) isolates of M. bovis were positive for ELISA by using bovine PPD as coating antigens. Out of 18 tuberculin reactors showing NVL, 2 (11.1%) isolates of M. bovis could be isolated from them and zero (0%) serum samples were positive by ELISA by using bovine PPD as coating antigen.
The results obtained in the current study agreed with those reported by [62, 63] who stated that, antibody response to M. bovis infection is certainly not uniform. This phenomenon was previously suggested in serological analysis by [64] who studied the humoral immune response to M. bovis infection in cattle and concluded that, it was characterized by highly heterogeneous antigen recognition. It is concluded that single intradermal tuberculin test, culture and isolation of the mycobacterium are recommended for determination of a true prevalence of bTB in the herd to be established.
The recent lateral flow rapid kits could be used for initial tuberculosis screening in combination with tuberculin skin test (TST) for improving sensitivity of bovine tuberculosis screening, thereby leading to more successful control programs in developing countries. The rapid test is proposed as a potentially useful ancillary assay for bTB. In addition, it may be most suitable for surveillance, especially if an immediate result is needed. The differences in the sensitivity and specificity of the different LFA kits sources available in the market necessitate that, the importance of choosing type of diagnostic kits used for rapid detection of bovine tuberculosis. Also, these kits alone may be not enough for accurate diagnosis of bovine tuberculosis.