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Results of tuberculin test, isolation, simplex real-time PCR using atpE, multiplex real-time PCR using lpqT and RD1, multiplex conventional PCR targeting RD1, RD4, RD9, and RD12, and simplex conventional PCR targeting RD4, and RD9
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Mycobacterium tuberculosis complex (MTBC) has the potential to cause infections in animals and human beings. The combination of real-time PCR targeting atpE or lpqT and RD1, and conventional PCR targeting regions of difference (RD) was rigorously evaluated as a descriptive molecular epidemiology tool. A total of 2100 cattle and buffaloes from the M...
Citations
... To amplify the mpb70/m22 and regions of difference RD9-RD4 genes, nested and endpoint PCRs were performed respectively. The first one allows identifying members of the Mycobacterium tuberculosis complex, and the second to differentiate M. bovis (Elsayed and Amer, 2019;Carrisoza-Urbina et al., 2019). According to the manufacturer's specifications, a commercial PCR Master Mix 2x kit (Thermo Scientific) was used. ...
... Although M. tb primarily causes TB in humans; however, it is also occasionally isolated from diseased cattle and buffaloes in several countries of Asia and Africa [6][7][8][9][10]. M. bovis also exhibits an extensive range of hosts, encompassing domestic animals, livestock, wildlife, and humans [11]. ...
... In the absence of clinical signs in buffalo, the difficulty of isolating M. bovis from live animals, and low antibody titers during the early phase of infection, the effectiveness of clinical, bacteriological, and serological diagnostic tests is limited. Alternative diagnostic methods have been proposed, including serological ELISA tests and molecular diagnostic tests using polymerase chain reaction (PCR) [9]. ...
... Preclinical stages of tuberculosis can be diagnosed in live animals using tests that measure cell-mediated immunity, such as the intradermal injection of tuberculin, the IFN-γ test [10], and lymphocyte transformation tests. Additionally, some serological methods evaluate humoral immune responses, such as ELISA tests [11], microbiological culture methods, and molecular tests [9]. The key to the efficiency and accuracy of these diagnostic methods lies in the balance between sensitivity and specificity. ...
Introduction: Bovine tuberculosis (bTB) is a zoonotic infectious disease present in Colombia, caused by Mycobacterium bovis, and causes tuberculosis in water buffalo (Bubalus bubalis). Diagnosis of bovine tuberculosis through the intradermal test is difficult; evaluating and understanding the behavior of other diagnostic tests is necessary. Objective: To describe the behavior and results of different diagnostic methods for bovine tuberculosis in water buffalo positive for the Purifed Proteic Derivate (DPP) intradermal test. Methodology: In water buffaloes positive for comparative cervical tuberculin test, different diagnostic methods were applied, described, and compared: Ziehl-Neelsen staining, microbiological culture, histopathological analysis, and PCR-HRM. Results: Histopathological tests showed that 26 water buffalo positive for DPP (52%) had histological lesions compatible with bovine tuberculosis. 37% of the evaluated samples from tuberculin-positive Buffalo's lungs and secondary lymph nodes showed acid-alcohol-resistant bacillus with Ziehl-Neelsen staining. Four samples of Mycobacterium bovis from tuberculin-positive buffalo were isolated and identified, with two of these isolates confirmed from tissues with PCR-HRM, and three buffalo with microbiological isolates presented granulomatous lesions through histological analysis. Seventeen tuberculin-positive buffalo (34%) tested positive for real-time PCR HRM, and nine of these buffalo did not have histological lesions compatible with bTB and were confirmed with the molecular test. Conclusion: Our results provide positive evidence of histological findings, microbiological isolation, and molecular diagnosis of tuberculin-positive water buffalo in the lowlands of Colombia. None of the complementary tests performed showed 100% concordance with the comparative cervical tuberculin test results for bTB.
... According to current legislation (64/432/ECC), the official tests for determining the health status of herds are the intradermal tuberculin test, and the microbiological examination of bovine tissues [ 17 ]. However, the skin tuberculin test often causes false-positive reactions in cattle, buffalo and other animals due to interference from non-tuberculous mycobacteria [ 18 ]. Although a bacteriological test is the "gold standard" for the detection of tuberculosis in slaughtered animals, other methods for detecting MTBC members directly from tissue samples have been developed in the last decade to accelerate the diagnosis. ...
... Although a bacteriological test is the "gold standard" for the detection of tuberculosis in slaughtered animals, other methods for detecting MTBC members directly from tissue samples have been developed in the last decade to accelerate the diagnosis. Different RT-PCR studies showed that the real-time PCR and conventional PCR targeting the RDs regions of the genome are fast and accurate methods comparable to culture detection and have great potential for detecting infected live animals and confirming cases in slaughtered animals [ 18 ]. Our results from qPCR in the studied samples are completely comparable to the results obtained from the bacteriological test. ...
... Our results from qPCR in the studied samples are completely comparable to the results obtained from the bacteriological test. Other authors have reported similar results and demonstrated the usefulness of qPCR in diagnostic laboratory practice, even if it cannot be used for differentiation between members of the MBTC [17][18][19]. ...
Representatives of Mycobacterium tuberculosis complex (MTBC) are potential causative agents of tuberculosis in animals and humans, and Mycobacterium bovis is considered a major one among domestic and wild ruminants. In the last twenty years, the role of another representative of MTBC – Mycobacterium caprae, has been proven in the countries of Central and Southern Europe. Study sample included 121 diagnostic materials from lymph nodes and lungs from cattle, positively and doubtfully PPD tuberculin-reacted, originating from 6 farms belonging to the 4 regions in Northern and Southern Bulgaria. The bacteriological examination showed typical growth for mycobacteria for 110 (90.9%) samples, which was confirmed by qPCR. By RD4-PCR we proved that 102 (92.7%) of the mycobacterial strains were M. caprae and the remaining 8 strains (7.3%) were M. bovis. This defines M. caprae as a dominant species in the etiology of tuberculosis in cattle in Bulgaria. In conclusion, we confirmed that the application of real-time PCR is an accurate and convenient method for rapid detection of M. bovis from cattle in the Bulgarian settings. RD4-PCR provides a sufficiently high differentiation and can be used for first-line typing of M. bovis isolates in Bulgaria. This proves the need for the simultaneous application of both methods in the diagnosis of bovine tuberculosis in Bulgaria.
... Furthermore, the tuberculous lesions in cattle and buffalo carcasses represented 0.7 and 0.45%, respectively from El-Mahalla El-Kubra abattoir, Mid-Delta of Egypt at 2016 [10]. In April 2019, a prevalence of 3.5% tuberculinpositive cases was declared by Elsayed and Amer [11], from cattle and Egyptian water buffaloes in Menoufia, Sharkia, Gharbia, Dakahlia, Elbuhaira, and Cairo. During August 2019, a prevalence of 3.43% was confirmed in 27 dairy herds from; Alexandria, Beheira, Daqahlia, Gharbia, Kafr EL Sheikh, Menoufia, and Sharkia [12]. ...
... We found a prevalence of 1.2 and 1.6% tuberculinpositive cases for buffaloes and cattle, respectively, with an overall rate of 1.5%. This is nearly similar to published reports from Egypt 2016 [9], and lower than the published data at the beginning of 2019 [11] and the mid of 2019 [35]. This fluctuation of records may be due to the different times of animal testing and the strict implementation of test and slaughter policy adopted by the General Authority for Veterinary Services for controlling mycobacterium infections in animals and potentially spreading to humans [3]. ...
... The implementation of real-time PCR using atpE primer/probe proved that all the DNA from the tissue samples and isolates harbored mycobacteria. These results agree with the published records from Egypt [11]. The high detection results of the real-time PCR using atpE primer/probe were based on the ability of this realtime PCR technique to quantify very few mycobacterial DNA copies in the tissue samples and isolates [22,44]. ...
Background: Mycobacterium bovis notoriously causes detrimental infections in bovines and humans. In this study,
1500 buffaloes and 2200 cattle were tested by single intradermal comparative cervical tuberculin test and compared
with the detection rates of M. bovis isolation, real-time and simplex PCR, and flow Cytometry.
Results: The tuberculin test is the reference test in Egypt, the positive rate was 54/3700 (1.5%) composed of 18/1500
(1.2%) buffaloes and 36/2200 (1.6%) cattle which were mandatorily slaughtered under the Egyptian legislation, after
postmortem examination the non-visible-lesion proportion was 39/54 (72.2%) which surpassed the visible-lesion rate
15/54 (27.8%) with (p < 0.0001). The samples from each case were pooled into one sample representing the case, and
the isolation rate of M. bovis was 25/54 (46.3%). Real-time PCR using atpE was positive for mycobacteria on the genus
level in 18/18 (100%) and 5/5 (100%) of tissue samples and isolates, respectively; simplex PCR detected M. bovis in
44/54 (81.5%) and 25/25 (100%) of tissue samples and isolates, respectively. Flow Cytometry evaluation of the CD4+,
CD8+,
WC1+
δγ, and CD2+
cell phenotypes showed increased counts in the tuberculin-positive cases compared with
negative cases (p < 0.0001), and these phenotypes in the tuberculin-positive cases increased after antigen stimulation
than in the negative cases (p < 0.0001). Detection rates of PCR techniques and flow Cytometry exceeded that of
bacterial isolation (p < 0.0001) and exhibited a strong correlation.
Conclusions: The skin test suffers from interference from non-tuberculous mycobacteria able to cause false-positive
reactions in cattle and other species. Real-time PCR using atpE, conventional PCR targeting RDs, and flow Cytometry
are rapid and accurate methods that correlate with the isolation and can be promising for detection and confirmation
of infected live and slaughtered cases.
Keywords: Cattle and buffalo, Conventional PCR, Flow cytometry, Mycobacterium bovis, Real-time PCR atpE
... PCR techniques are widely used for the diagnosis of bTB and have several advantages; they are quick, applied within a few hours which means rapid diagnosis and efficient control, overcome the lack of specificity of other traditional tests such as histopathology, and are able to identify the mycobacteria either from culture or clinical specimens [68,103]. Moreover, several PCR techniques targeting numerous genes and regions can be used for differentiation between members of MBTC, such as ESAT-6 and CFP-10, which are protein products of esxA and esxB genes, respectively [104], atpE and lpqT [1], regions of difference ( RD 1,4,9,12) [1,105], RvD1-Rv2031c [76], and insertion sequences (IS) as IS1081 [80] and IS6110. Numerous studies targeted the insertion sequence region IS6110 as it is found in all members of MBTC [103,104,106]. ...
... PCR techniques are widely used for the diagnosis of bTB and have several advantages; they are quick, applied within a few hours which means rapid diagnosis and efficient control, overcome the lack of specificity of other traditional tests such as histopathology, and are able to identify the mycobacteria either from culture or clinical specimens [68,103]. Moreover, several PCR techniques targeting numerous genes and regions can be used for differentiation between members of MBTC, such as ESAT-6 and CFP-10, which are protein products of esxA and esxB genes, respectively [104], atpE and lpqT [1], regions of difference ( RD 1,4,9,12) [1,105], RvD1-Rv2031c [76], and insertion sequences (IS) as IS1081 [80] and IS6110. Numerous studies targeted the insertion sequence region IS6110 as it is found in all members of MBTC [103,104,106]. ...
... In addition, Algammal et al. reported that RT-PCR is the most sensitive rapid diagnostic test for detection of M. bovis from tissues and provides higher positive values than culturing [68]. However, other studies confirmed the usefulness of combination between RT-PCR and conventional PCR for rapid identification and discrimination between members of MBTC [1]. ...
Bovine tuberculosis is a serious infectious disease affecting a wide range of domesticated and wild animals, representing a worldwide economic and public health burden. The disease is caused by Mycobacterium bovis and infrequently by other pathogenic mycobacteria. The problem of bovine tuberculosis is complicated when the infection is associated with multidrug and extensively drug resistant M. bovis. Many techniques are used for early diagnosis of bovine tuberculosis, either being antemortem or postmortem, each with its diagnostic merits as well as limitations. Ante-mortem techniques depend either on cellular or on humoral immune responses, while postmortem diagnosis depends on adequate visual inspection, palpation, and subsequent diagnostic procedures such as bacterial isolation, characteristic histopathology, and PCR to reach the final diagnosis. Recently , sequencing and bioinformatics tools have gained increasing importance for the diagnosis of bovine tuberculosis, including, but not limited to typing, detection of mutations, phylogenetic analysis , molecular epidemiology, and interactions occurring within the causative mycobacteria. Consequently , the current review includes consideration of bovine tuberculosis as a disease, conventional and recent diagnostic methods, and the emergence of MDR-Mycobacterium species.
... Furthermore, the tuberculous lesions in cattle and buffalo carcasses represented 0.7 and 0.45%, respectively from El-Mahalla El-Kubra abattoir, Mid-Delta of Egypt at 2016 [10]. In April 2019, a prevalence of 3.5% tuberculinpositive cases was declared by Elsayed and Amer [11], from cattle and Egyptian water buffaloes in Menoufia, Sharkia, Gharbia, Dakahlia, Elbuhaira, and Cairo. During August 2019, a prevalence of 3.43% was confirmed in 27 dairy herds from; Alexandria, Beheira, Daqahlia, Gharbia, Kafr EL Sheikh, Menoufia, and Sharkia [12]. ...
... We found a prevalence of 1.2 and 1.6% tuberculinpositive cases for buffaloes and cattle, respectively, with an overall rate of 1.5%. This is nearly similar to published reports from Egypt 2016 [9], and lower than the published data at the beginning of 2019 [11] and the mid of 2019 [35]. This fluctuation of records may be due to the different times of animal testing and the strict implementation of test and slaughter policy adopted by the General Authority for Veterinary Services for controlling mycobacterium infections in animals and potentially spreading to humans [3]. ...
... The implementation of real-time PCR using atpE primer/probe proved that all the DNA from the tissue samples and isolates harbored mycobacteria. These results agree with the published records from Egypt [11]. The high detection results of the real-time PCR using atpE primer/probe were based on the ability of this realtime PCR technique to quantify very few mycobacterial DNA copies in the tissue samples and isolates [22,44]. ...
Background
Mycobacterium bovis notoriously causes detrimental infections in bovines and humans. In this study, 1500 buffaloes and 2200 cattle were tested by single intradermal comparative cervical tuberculin test and compared with the detection rates of M. bovis isolation, real-time and simplex PCR, and flow Cytometry.
Results
The tuberculin test is the reference test in Egypt, the positive rate was 54/3700 (1.5%) composed of 18/1500 (1.2%) buffaloes and 36/2200 (1.6%) cattle which were mandatorily slaughtered under the Egyptian legislation, after postmortem examination the non-visible-lesion proportion was 39/54 (72.2%) which surpassed the visible-lesion rate 15/54 (27.8%) with (p < 0.0001). The samples from each case were pooled into one sample representing the case, and the isolation rate of M. bovis was 25/54 (46.3%). Real-time PCR using atpE was positive for mycobacteria on the genus level in 18/18 (100%) and 5/5 (100%) of tissue samples and isolates, respectively; simplex PCR detected M. bovis in 44/54 (81.5%) and 25/25 (100%) of tissue samples and isolates, respectively. Flow Cytometry evaluation of the CD4⁺, CD8⁺, WC1⁺δγ, and CD2⁺ cell phenotypes showed increased counts in the tuberculin-positive cases compared with negative cases (p < 0.0001), and these phenotypes in the tuberculin-positive cases increased after antigen stimulation than in the negative cases (p < 0.0001). Detection rates of PCR techniques and flow Cytometry exceeded that of bacterial isolation (p < 0.0001) and exhibited a strong correlation.
Conclusions
The skin test suffers from interference from non-tuberculous mycobacteria able to cause false-positive reactions in cattle and other species. Real-time PCR using atpE, conventional PCR targeting RDs, and flow Cytometry are rapid and accurate methods that correlate with the isolation and can be promising for detection and confirmation of infected live and slaughtered cases.
... The use of techniques and methods involving molecular biology, such as Polymerase Chain Reaction (PCR), has reduced the time required for the diagnosis of bTB. Several studies demonstrate the use of PCR in complementing the identification of M. bovis (Zumárraga et al. 2012, Barandiaran et al. 2019, Fico et al. 2019, such as the work of Elsayed & Amer (2019), who used PCR directly from tissue samples, demonstrating the technique's speed compared to cultivation for species identification. Sales et al. (2014) compared different primers for the detection of M. bovis, proving that Mb.400 primers, targeting fragments flanking the region of differentiation 4 (RD4), are the ones that demonstrated the best results in the PCR for bTB. ...
Bovine tuberculosis (bTB) is an infectious disease caused by Mycobacterium bovis, affecting domestic animals, wild animals and humans. In captivity, for wild animals, bTB represents a risk to animal keepers and zoo visitors, in addition to the possibility of spreading the infection to domestic animals or through the trade of infected wild animals. Sambar (Cervus unicolor), red deer (Cervus elaphus) and fallow deer (Dama dama) from a safari park in the State of Rio Grande do Sul, Brazil, showed a clinical condition of dyspnea and weight loss. Some animals died and showed lesions suggestive of tuberculosis (LST), which were confirmed by histopathology. After the interdiction of the safari park by the state veterinary authorities, 281 deer were euthanized with the authorization of the “Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis” (IBAMA). Retropharyngeal and submandibular lymph nodes and viscera were collected from 21 animals, which were grown in Stonebrink medium for up to 90 days. After DNA extraction from the bacterial colonies, PCR was performed for targets flanking the region of differentiation 4 (RD4). Of the 21 samples, 14 (66.7%) presented LST with a granulomatous appearance, a whitish coloration, and caseous or calcified consistency, and seven samples (33.3%), showed no lesions. In the culture of 14 samples with LST, 13 (92.8%) presented bacterial growth compatible with M. bovis. In the cultivation of the seven samples without LST, four (57.1%) presented colonies compatible with M. bovis. PCR and DNA sequencing of the PCR amplicons detected as positive all the 17 (100%) bacteriological cultures suggestive of M. bovis, thus confirming the outbreak of bTB in deer. Decisions about positive tested and suspicious animals should be taken based on the evaluation of the risk of transmission to the rest of the zoological animals, animal welfare, conservation considerations and, the zoonotic potential of this pathogen.
The Slaughter house is the place in which the animals are slaughtered for human consumption. The Slaughter house plays important role in prevention of zoonotic diseases between animals and humans like Mycobacterium tuberculosis as reemerging foodborne illness and also prevent infectious diseases between animals. Bovine Mycobacterium tuberculosis is caused by a species of pathogenic Gram positive, acid fast stain bacteria in the Mycobacteriaceae family. the causative agent bacteria of Bovine tuberculosis as reemerging foodborne illness tuberculosis bacteria has an waxy cover on its surface primarily due to the presence of acid called mycolic which refers the cells impervious to Gram staining, and as a result, the causative agent bacteria of Bovine tuberculosis as reemerging foodborne illness may appear weakly Gram-positive. Acid-fast bacilli by using certain stains called Ziehl Nielsen, or through using stain called fluorescent such as aura mine are used to identify the cause of Bovine tuberculosis as reemerging foodborne illness with a microscope. The Bacteria cause Bovine tuberculosis as a reemerging foodborne illness is aerobic bacteria and needs high concentrations of oxygen. Mainly this bacteria is pathogenic to human and mammal's respiratory system, it infects the lungs. The most diagnostic means for Bovine tuberculosis as a reemerging foodborne illness are the tuberculin skin examination, stain of acid-fast, laboratory culture, and through using polymerase chain reaction method.
Abattoir is the place in which the animals are slaughtered for human consumption. Abattoir plays important role in
prevention of zoonotic diseases between animals and humans like Mycobacterium tuberculosis as reemerging
foodborne disease and also prevent infectious diseases betwwen animals. Mycobacterium tuberculosis is caused by
a species of pathogenic bacteria in the family Mycobacteriaceae . the causative agent bacteria of Bovine tuberculosis
as reemerging foodborne disease tuberculosis bacteria have an unusual, waxy coating on its cell surface primarily
due to the presence of mycolic acid. This coating makes the cells impervious to Gram staining, and as a result, the
causative agent bacteria of Bovine tuberculosis as reemerging foodborne disease can appear weakly Gram-positive.
Acid-fast stains such as Ziehl–Neelsen stain, or fluorescent stains such as auramine are used instead to identify the
causative agent of Bovine tuberculosis as reemerging foodborne disease with a microscope. The causative agent
bacteria of Bovine tuberculosis as reemerging foodborne disease are highly aerobic and requires high levels of
oxygen. Primarily a pathogen of the mammalian respiratory system, it infects the lungs. The most frequently used
diagnostic methods for Bovine tuberculosis as reemerging foodborne disease are the tuberculin skin examination ,
acid-fast stain, culture, and polymerase chain reaction
