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Challenges in diagnosing bovine tuberculosis through surveillance and characterization of Mycobacterium species in slaughtered cattle in Kolkata

BMC Veterinary Research volume 20, Article number: 478 (2024) Cite this article

Abstract

Background

Tuberculosis in cattle is caused by Mycobacterium tuberculosis complex (MTBC) species. Apart from MTBC, different Nontuberculous Mycobacteria (NTM) species have also been isolated from cattle. The presence of NTM infection in bovines makes the diagnosis of bovine tuberculosis (bTB) a cumbersome task. Therefore, a cross sectional study was conducted to isolate and characterize different Mycobacterium spp. from a slaughterhouse situated in Kolkata, a city in the eastern part of India.

Results

Out of 258 morbid samples, 98 isolates were found to be positive for bacterial growth, and 35% (n = 34) were positive for Mycobacterium. 94% of Mycobacterial cultural isolates were NTM (n = 32), and the rest (n = 2) were found to be MTBC. Species-level identification of the isolates by hsp65 sequencing revealed that out of 32 isolates, 24 were M. fortuitum, three were M. abscessus, two each were M. chelonae and M. parascrofulaceum, and one was M. novocastrense. A phylogenetic tree with partial hsp65 gene sequences was also constructed to determine the relatedness of the unknown isolates to the reference strains.

Conclusion

Both NTM species and MTBCs were identified from TB-like lesions in cattle that were slaughtered at the Kolkata abattoir. This discovery may indicate that NTM contributes to the development of lesions in cattle. Also, we recommend implication of more specific diagnostic tests for bTB.

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Background

Bovine tuberculosis (bTB) is a chronic infectious disease that has been widely documented in cattle and has high potential for zoonotic transmission. bTB mostly affects the lungs, the lymph nodes, and other organs, depending on the route of infection [1,2,3]. bTB is caused by members of the Mycobacterium tuberculosis complex (MTBC), primarily Mycobacterium bovis. There have been significant differences in the prevalence of bTB in dairy cattle over the past ten years, with reports from various regions of India indicating prevalence rates ranging from 4.48 to 28.8% (Reviewed extensively by Refaya et al., 2020) [4]. Four recent studies on bTB from three states from eastern part of the country, Assam, Guwahati, Meghalaya and West Bengal, reported 28% [5], 10.55% [6], 12.89% [7], 21.4% [8], respectively. All these reports on bTB incidence in Indian bovine populations from different states were based on conventional diagnostic techniques such as single intradermal test (SIT), single intradermal comparative tuberculin test (SICT), double intradermal test (DIT), enzyme-linked immunosorbent assay (ELISA), interferon-gamma release assay (IGRA), Ziehl-Neelsen (ZN) staining and detailed post-mortem (PM) examinations [9].

In addition to MTBC, other Mycobacterial species, collectively referred to as nontuberculous mycobacteria (NTM), also play a significant role as a source of infections in humans and animals. Among approximately 200 recognized NTM species, 30 are reported to be pathogenic to both animals [10] and humans [11]. The importance of NTM as an opportunistic pathogen in animals is also growing [12], as livestock as well as wild animals are naturally exposed to NTM, and the tendency for infection, particularly in grazing-free cattle, is high [13, 14]. Due to the lack of intensive knowledge about the mode of transmission of diverse NTM species, these ubiquitous bacterial populations are plausible zoonotic threats that cause large economic burdens on humans and livestock. Another problem caused by NTM infections in cattle is the interference of certain NTM species with the diagnosis of bTB [15,16,17,18,19,20]. The presence of NTM in cattle is also reported to negatively impact vaccination [21].

Recently, highly sensitive and specific nucleic acid amplification (NAAT) techniques have been widely used for the rapid detection and differentiation of MTBC and NTM [14]. A study conducted on a farm situated in the northern part of India reported the presence of NTM species such as M. kansasii, M. intracellulare and M. vaccae in the trans-tracheal washes of cattle and buffaloes [22]. To date, however, no investigation has been performed to pinpoint the specific Mycobacterial species occurring in tuberculous-like lesions in cattle in India. Therefore, the present study was performed to identify the Mycobacterial species present in the suspected TB lesions in slaughtered cattle in eastern part of India.

Results

Sampling, macroscopic examination, Z-N staining and culture

In this study, a total of 1020 slaughtered cattle were examined macroscopically. Variable-sized nodular lesions (Fig. 1) were detected in the organs of 186/1020 (18.24%) cattle. In cattle, a tuberculous-like nodule in the organs is usually characterized by a yellowish-white or grayish-white granuloma encased in a capsule of varying thickness. In total, 258 morbid samples were collected from the slaughterhouse, of which 13/258 (5.03%) lung samples and 7/258 (2.71%) liver samples were found to be Z-N positive by direct screening via the Z-N staining method. However, bacterial growth (Fig. 2) was recorded in 98/258 (38%) samples (lungs = 53, liver = 45).

Fig. 1
figure 1

Typical tuberculous-like lesions observed macroscopically in (a) lung and (b) liver. Calcified area marked with arrows

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Fig. 2
figure 2

Cultural isolation of MTBC and NTM in Lowenstein-Jensen media. Colonies of (a) MTBC, (b-d) NTM

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Direct screening of isolates via duplex PCR

Duplex PCR with primers spanning the Mycobacterial 16S rRNA and MPB 70 gene fragments was used to identify Mycobacterium species and to distinguish MTBC and NTM species. Amplification of the 1030 bp fragment from 16S rRNA confirmed that the species was Mycobacterium sp., whereas co-amplification of the 1030 bp and 372 bp fragments indicated that the species was an MTBC. Out of 98 bacterial growth, 34 isolates were found to yield a 1030 bp product. In two isolates, both 1030 bp and 372 bp products were amplified, indicating that they were members of the MTBC family, and 32 isolates were negative for the MPB 70 gene fragment (Fig. 3). Of the 34 isolates, 10 originated from crossbred cattle, while 24 were from indigenous cattle (Table 1).

Table 1 Different tissue samples screened for the presence of Mycobacterium spp., number of positive samples diagnosed by PCR-based diagnostic tests and breed distribution of positive animals
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Fig. 3
figure 3

Agarose gel electrophoresis for screening of MTBC and NTM isolates by duplex PCR. Lanes showing amplification of only 1030 bp products are Mycobacterium species. Lanes showing the amplification of both the 1030 bp and 372 bp (MTBC-specific) products are MTBC species. M1: 100 bp Ladder; L2 - L10: Unknown Samples, L1 & L11: M. tuberculosis H37Rv, L12: M. bovis AN5, L13: M. orygis, L14: M. fortuitum, L15: M. smegmatis, L16: M. kansasii, L17: M. terrae, L18: NTC, M2: 1000 bp Ladder

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Species-level confirmation of Mycobacteria

Species-level detection of NTM species is widely performed by amplification and sequencing of the 439 bp hsp65 gene fragment as described earlier by Telenti et al. (1993) [23]. All 34 isolates were positive for hsp65 (Fig. 4). Among 34 Mycobacterial isolates, two were detected as MTBC, and 32 were detected as NTM. Out of 32 NTM, 24 (75%) were identified as M. fortuitum, 3 (9.3%) as M. abscessus, 2 (6.25%) each as M. parascrofulaceum and M. chelonae and one (3.12%) as M. novocastrense. Two MTBC isolates were identified as M. orygis matching the RD-real time PCR pattern with the signature patterns listed in Table 2. Amplification plots and corresponding Cq values of two MTBC isolates and M. tuberculosis H37Rv (positive controls) have been represented in Fig. 5. The numbers of MTBCs and different NTM species are shown in Fig. 6.

Table 2 Signature patterns of RD real-time PCR results used to determine MTBC species
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Fig. 4
figure 4

Agarose gel electrophoresis showing amplification of the 439 bp hsp65 gene fragment in Mycobacterium species. L1-L7: representative hsp65-positive lung samples; L8-L14: representative hsp65-positive liver samples; L15-L17: reference strains of M. fortuitum, M. kansasii, and M. tuberculosis H37Rv were used as positive controls; L18: negative control

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Fig. 5
figure 5

Amplification plots depicting presence or absence of RD1, RD4, Ext-RD9, RD12 and Rv0444c in MTBC-RD real-time PCR. (a-d) MTBC isolate 1 and isolate 2 showing positive amplification of RD1, Ext-RD9 and Rv0444c. This indicates the species of two MTBC isolates as M. orygis. (e-f) M. tuberculosis H37Rv strain was used as positive control. This shows positive amplification of RD1, RD4, Ext-RD9 and RD12

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Fig. 6
figure 6

Distribution of different NTM and MTBC species isolated from slaughterhouse samples. NTM species were identified by sequencing partially amplified hsp65 gene and homology search by BLASTn. MTBC isolates were confirmed as M. orygis by RD Real-time PCR

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Phylogenetic analysis

Ancestral states were inferred using the neighbour-joining (N-J) method [24] and the Tamura-Nei model [25] with 1000 bootstrap replicates. The hsp65 gene sequences of reference strains from NCBI [M. abscessus (accession no: JX154122.1), M. chelonae (accession no: JX154110.1) and M. fortuitum (accession no: JX154102.1) and M. orygis (Coordinates 530415 to 530855 of M. orygis strain MUHC/MB/EPTB/Orygis/51145 chromosome, complete genome, accession number: CP063804.2)] were included in the phylogenetic analysis to ensure the relatedness of the Mycobacterial isolates of slaughterhouse origin to the reference strains. As shown in Fig. 7, isolate nos. 138 and 139 (blue nodes) exhibited strong phylogenetic relationships with the M. chelonae reference strain gene sequence. Similarly, isolate no. 140 (green node) showed a phylogenetic relationship with the M. abscessus reference sequence. Two MTBC isolates (Isolate nos. 115_1 and 117 with red nodes) were found to have distinct cluster positions in the rooted phylogenetic tree, whereas the rest of the isolates (violet nodes) were phylogenetically similar to hsp65 in the M. fortuitum reference sequence.

Fig. 7
figure 7

hsp65-based phylogenetic relationships of different Mycobacterium spp. isolates determined by the N-J method using MEGA version 7 with 1000 bootstrap replicates

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Discussion

The present study on slaughterhouse surveillance focused on estimating the prevalence of Mycobacterium spp. in cattle and identifying the Mycobacterial species circulating in eastern India. The prevalence rate of Mycobacterium spp. was found to be greater in indigenous cattle. The breed-wise prevalence of bTB has been reported by various researchers [26,27,28]. In a slaughterhouse study, Sharma et al. (1985) [26] concluded that Indian breeds were less affected by bTB than were pure European breeds such as Jersey, Holstein Friesians, and Brown-Swiss. Thakur et al. also reported greater positivity in Jersey (30%) and Jersey-cross (45.5%) breeds in Himachal Pradesh [28]. Similarly, a greater prevalence of tuberculosis in the exotic breed and their crosses than in indigenous cattle was reported by Das et al., (2018) (34.6% vs. 10.5%) [8] and Sripad et al., (2018) (2.66% in Holstein Friesians and 1.37% in Jersey vs. 0% in indigenous cattle) [29]. However, no study has yet been conducted on the prevalence of NTM in animals in India. In the present study, 5.88% (n = 2) of the Mycobacterium isolates were confirmed to be MTBC, and the remaining 94.12% (n = 32) were confirmed to be NTM, and the susceptibility of cattle to NTM was found to be much greater in indigenous cattle.

We report the presence of MTBC in slaughtered bovine in eastern India, which may pose public health threat. hsp65 sequencing, MTBC-RD-Real time PCR assay and phylogenetic analysis revealed the relatedness of the isolates (115_1 and 117) with M. orygis. M. orygis has become a zoonotic hazard in south Asia [30] with recent occurrences of multispecies cases involving humans, dairy cattle, and wild ungulates reported from India [30,31,32,33,34,35]. Research from Bangladesh and Nepal has also shown that M. orygis is present in livestock and free-ranging wild animals. The identification of M. orygis in ten human patients in south Asia [33] and reports of the infection spreading from an Indian farm labourer to cattle in New Zealand [35] suggest endemicity in the area, underscoring the critical need for molecular epidemiological surveillance study.

The isolation and species-level confirmation of the NTM (> 90%) reveal that a substantial percentage of the NTM infection other than MTBC is circulated in cattle. This remains a matter of concern for epidemiologists and public health veterinarians, as NTM causing clinical disease have been identified and, in many geographical regions, causes a greater disease burden than TB [36]. NTM are opportunistic environmental pathogens that can infect both humans and animals. They pose a significant threat to public health, especially for those with acquired immune deficiency syndromes. According to recent scientific research, NTM are no longer just considered to be common environmental organisms, but also potentially harmful pathogens that can affect humans and animals [10]. Furthermore, some NTM species, such as M. fortuitum, M. abscessus, and M. novocastrense, have been reported to cause human infections [37]. Current research highlights the necessity for public health awareness anticipating the possibility of these NTM pathogens being transferred from animal carcasses to workers in slaughterhouses. In animals, a variety of NTM species induce broadly cross-reactive anti-Mycobacterial immune responses to which livestock species are naturally exposed. These immune responses interfere with current standard diagnostic assays, viz., tuberculin tests and interferon gamma assays [18, 38]. The use of purified protein derivatives, a crude mixture of proteins that may contain epitopes common in NTM as well as MTBC, including Mycobacterium bovis, is thought to be the cause of such cross-reactive immune responses [18]. Furthermore, exposure to NTM may reduce the efficacy of the M. bovis BCG vaccine [39, 40]. The current study revealed a greater prevalence of NTM among cattle in eastern India, which likely interferes with the diagnosis of bTB by in vivo and in vitro tests such as tuberculin and gamma interferon assays.

Furthermore, in the present study, 70.59% (n = 24) of the isolates were under the M. fortuitum. In humans, M. fortuitum is frequently associated with skin, soft tissue, and bone infections but rarely causes pulmonary disease, except in cases of lipoid pneumonia, gastroesophageal disorders, or disseminated diseases [41]. The higher prevalence of the M. fortuitum in animal tissues has attracted increased amounts of attention for detailed studies on the pathogenesis of this group of organisms in animals [37].

Conclusion

In this study, both of MTBC and NTM species were isolated from tuberculous-like lesions of cattle slaughtered. However, information on the transmission dynamics of Mycobacterium species in cattle, including samples from environmental sources such as water and soil, is required to understand the hazards and associated risk factors. The presence of various NTM strains in cattle herds in the eastern part of India should be considered while carrying out bTB surveillance programs. We recommend the use of advanced molecular techniques such as whole-genome sequencing for in-depth characterization of the isolates. Moreover, nationwide screening and ongoing surveillance under the One Health strategy should be carried out in accordance with the World Health Organization’s End TB Strategy to tackle this deadly zoonotic infection.

Methods

Study site

The study was performed in cattle slaughtered at the Kolkata slaughterhouse, which is located in Kolkata city of West Bengal state, India. Currently, this is the only licenced slaughterhouse under the aegis of Kolkata Municipal Corporation, where both nondescript and crossbred cattle brought mostly from peri-urban areas of neighbouring states and adjacent districts of West Bengal are slaughtered.

Sample collection

A total of 1020 cattle slaughtered between August 2019 and March 2020 were methodically inspected for tuberculous-like lesions in the parotid, mandibular, retropharyngeal, tonsil, left and right bronchial, cranial and caudal mediastinal, bronchial, tracheobronchial and mesenteric lymph nodes, and other organs, including the lungs, liver, spleen, kidney, peritoneum and pleural cavity. The lobes of the lungs were also visually examined and palpated to identify nodular lesions. In cattle, a tuberculous-like nodule in the organs is usually characterized by a yellowish-white or grayish-white granuloma encased in a capsule of varying thickness. Samples with variable-sized tuberculous-like nodular lesions were aseptically sliced (approximately 2 cm thick), transported to the laboratory while maintaining the cold chain as soon as possible and kept at − 20 °C until further analysis. This study did not require ethical clearance because it was conducted on slaughtered animals, and the examination of organs was a standard part of the monitoring process.

Sample processing

Each sample was processed following the standard protocol recommended by the WOAH, 2018 manual [42]. In brief, the tissue samples were manually chopped into smaller pieces and macerated using a mortar and pestle. An equal volume of 4% NaOH was added to the homogenate for decontamination, and the sample was centrifuged at 3000×g for 15 min. The sediment was neutralized with 2 N HCl using phenol red as an indicator. Finally, it was inoculated onto Lowenstein-Jensen (L-J) glycerol and L-J pyruvate solid media slants. The culture media were incubated at 37° C for 8 weeks, and growth was checked at regular intervals. If no visible growth was observed after the 8th week, the culture was considered negative. The impression smears of the tissue samples and the smears from the cultures were examined by using Ziehl-Neelsen (Z-N) smear microscopy for identification of acid-fast tubercle bacilli following a standard protocol [43]. Two loops of bacterial cells from colonies of probable positive growth cultures were mixed in 200 µl of distilled water and heat-killed at 85 °C for 45 min for subsequent molecular biology experiments.

Duplex PCR for differentiating MTBC and NTM species

DNA was isolated from the culture using a QIAampMini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Subsequently, duplex PCR with 16S rRNA and MPB 70 genes with amplicon sizes of 1030 bp and 372 bp, respectively, was employed to differentiate between the MTBC and NTM species [44].The PCR primer sequences spanning the 16S rRNA gene were MYCGEN Forward 5´-AGAGTTTGATCCTGGCTCA-3´ and MYCGEN Reverse 5´-TGCACACAGGCCACAAGGG-3. The primers used for the MPB70 gene were as follows: TB1 forward, 5´-GAACATCCGGAGTTGAC-3’; and TB1 reverse, 5’-AGCACGCTGTCAATCATGT-3’. The reaction mixture was prepared by mixing 0.1 µL each of four primers (10 pmol), 2.5 µL of 10× Taq buffer, 2 µL of 25 mM MgCl2, 200 µM dNTP mix and 1U of Taq polymerase enzyme (Thermo Fisher Scientific). PCR was carried out in a thermal cycler (Bio-Rad, USA), and the cycling conditions were as follows: initial denaturation at 94 °C for 5 min, followed by 39 cycles of denaturation at 94 °C for 30 s, annealing of primers at 62 °C for 3 min, extension at 75 °C for 3 min and a final extension at 75 °C for 7 min. In each run, DNA from M. tuberculosis H37Rv, M. bovis AN5, M. orygis and other NTM species was used as a positive control, and sterile water was used as a negative control. The PCR products were electrophoresed on a 1.5% agarose gel and visualized in a gel documentation system (Bio-Rad, USA).

Species-level identification of Mycobacterium spp. by hsp65 amplification and sequencing

PCR amplification of the partial hsp65 gene fragment and sequencing of the amplicons were performed to identify the particular species of Mycobacterium. The PCR sequences of primers used were as follows: forward (Tb11) 5´-ACCAACGATGGTGTGTCCAT-3´ and reverse (Tb12) 5´-CTTGTCGAACCGCATACCCT-3´, resulting in a 439 bp product [45]. The reaction mixture was prepared by mixing 0.5 µL of forward primer (10 pmol), 0.5 µL of reverse primer (10 pmol), 2.5 µL of 10X Taq buffer, 2 µL of 25 mM MgCl2, 200 µM dNTP mix and 1U of Taq polymerase enzyme (Thermo Fisher Scientific). PCR was carried out in a thermal cycler (Bio-Rad, USA), and the cycling conditions were as follows: initial denaturation at 96°C for 5 min, followed by 45 cycles of denaturation at 96°C for 40 s, annealing of primers at 60°C for 50 s, extension at 72°C for 1 min and a final extension at 72°C for 10 min. Standard cultures of the M. fortuitum, M. kansasii, and M. tuberculosis H37Rv strains (from ICMR-National Jalma Institute for Leprosy & other Mycobacterial Diseases) were included in the reaction as positive controls. The samples that were confirmed to be positive by hsp65 gene-specific PCR were Mycobacterium species. PCR products were sequenced by the Sanger sequencing method at Christian Medical College, Vellore, India. Briefly, the PCR products were cleaned by QIAquick purification Kit (Qiagen) as per the manufacturer’s instructions. The sequencing PCR reaction was carried out with 2 picomolar (0.2µM) concentrations of forward (Tb11–5´-ACCAACGATGGTGTGTCCAT-3´) and reverse primers (Tb12–5´-CTTGTCGAACCGCATACCCT-3’) along with the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA). PCR was performed with the initial denaturation of 1 min at 96 °C followed by 25 cycles of 10 s at 96 °C, 5 s at 50 °C, and 4 min at 62 °C in a Veriti 96-well thermal cycler. The fluorescently labelled sequencing PCR fragments were denatured, snap chilled and loaded into the 3500 Genetic Analyzer for capillary electrophoresis.

Species-level confirmation of MTBC by RD real-time PCR

A five-probe multiplex real-time PCR assay was performed to identify the species of two MTBC isolates, as described by Halse et al., (2011) [46] and Duffy et al. (2020) [33]. The assay detected presence of RD1, RD9, RD12, Rv0444c and a conserved region external to RD9 (Ext-RD9). The details of the primer, probe sequences and labelled reporter dye are presented in Table 3. This assay was carried out in a 20 µl volume using the TaqMan™ Multiplex Master Mix (Applied Biosystems, Vilnus, Lithuania). Each reaction mixture was prepared with 2X TaqManTM Multiplex Master Mix, 4 mM MgCl2, 450 nM forward and reverse primers, 125 nM probes (Table 3), sterile water, and 1 µl of DNA template. Thermocycling was performed in CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA) with the following cycling conditions: 1 cycle at 95 °C for 10 min, followed by 45 cycles at 95 °C for 15 s and 60 °C for 1 min. Fluorescence data acquisition, and data analysis were performed according to the manufacturer’s instructions. M. tuberculosis H37Rv was used as positive control.

Table 3 Primers, probes and labeled dye used for RD-Real-time PCR for species-level identification of MTBC isolates
Full size table

A positive target result with Cq value < 35 indicated the presence of the RD, whereas a negative target result with Cq value undetermined showed its absence. To ascertain the exact species of MTBC isolates the target patterns that resulted from RD PCR assay were compared to the signature patterns listed in Table 2.

Analysis of hsp65 sequences and phylogenetic analysis

Sequences of partial hsp65 gene fragment were assembled and aligned using BioEdit software program [47]. BLASTn was performed for each sequence, and more than 95% sequence similarity with the NTM species-specific hsp65 sequences in databases was considered to determine the species of the isolate. The hsp65 sequences of Mycobacterium species were aligned, and a phylogenetic tree was constructed with MEGA version 7 using the N-J method [24] and the Tamura-Nei model [25] with 1000 bootstrap replicates. The hsp65 gene sequences of reference strains, viz. M. abscessus (accession no: JX154122.1), M. chelonae (accession no: JX154110.1), M. parascrofulaceum (HM229795.1), M. novocastrense (HM807282.1) and M. fortuitum (accession no: JX154102.1) and M. orygis (Coordinates 530415 to 530855 of M. orygis strain MUHC/MB/EPTB/Orygis/51145 chromosome, complete genome, accession number: CP063804.2)] were included in the phylogenetic analysis to ensure the relatedness of the unknown isolates to the reference strains.

Data availability

All the data set(s) in support of the results of this article are comprised within the article.

Abbreviations

bTB:

Bovine tuberculosis

MTBC:

Mycobacterium tuberculosis complex

NTM:

Nontuberculous Mycobacteria

Z-N:

Ziehl-Neelsen

PCR:

Polymerase chain reaction

PM:

Postmortem

bp:

Base pair

BLASTn:

Nucleotide BLAST

N-J:

Neighbour-joining

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Acknowledgements

The authors are thankful to Vice Chancellor, West Bengal University of Animal and Fishery Sciences, Kolkata and Director, ICAR-Indian Veterinary Research Institute for undertaking the study. The authors express gratitude to Department of Biotechnology, Ministry of Science and Technology, Government of India for funding. Further, the assistance rendered by Kolkata Slaughter House Authority is also thankfully acknowledged.

Funding

The study was funded by Department of Biotechnology, Ministry of Science and Technology, Government of India for funding (Project ID: No. BT/ADV/Bovine Tuberculosis/2018).

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Authors and Affiliations

  1. West Bengal University of Animal and Fishery Sciences, 37 & 68, Kshudiram Bose Sarani, Kolkata, West Bengal, 700 037, India

    Molla Zakirul Haque, Chanchal Guha, Ayan Mukherjee, Partha Sarathi Jana, Ujjwal Biswas & Sangeeta Mandal

  2. ICAR-Indian Veterinary Research Institute, Eastern Regional Station, 37, Belgachia Road, Kolkata, West Bengal, 700 037, India

    Sukhen Samanta, Santanu Pal, Pramod Kumar Nanda, Samiran Bandyopadhyay, Arun K. Das & Premanshu Dandapat

  3. Christian Medical College, Vellore, Tamil Nadu, 632 004, India

    Manigandan Venkatesan & Joy Sarojini Michael

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  1. Molla Zakirul Haque
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Contributions

C.G. and P.D. designed the experiment and wrote the manuscript, M.Z.H. carried out the experiment, A.M. wrote the first draft of the manuscript, and analyzed the data, and C.G., P.S.J, U.B. revised the final version of the manuscript, M.Z.H., S.S., S.M. and S.P. contributed in sampling, data collection and revision of the manuscript; P.K.N., S.B. and A.K.D. analysed the data, M.V., J.S.M., contributed in DNA sequencing study. All authors have revised the final version of the manuscript.

Corresponding author

Correspondence to Premanshu Dandapat.

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As experimentation on live animals was not conducted in the study, no ethical approval was needed.

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Haque, M.Z., Guha, C., Mukherjee, A. et al. Challenges in diagnosing bovine tuberculosis through surveillance and characterization of Mycobacterium species in slaughtered cattle in Kolkata. BMC Vet Res 20, 478 (2024). https://doi.org/10.1186/s12917-024-04272-9

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