Mycobacterium bovis prevalence affects the performance of a commercial serological assay for bovine tuberculosis in African buffaloes

Van der Heijden, E.M.D.L. (Elisabeth); Cooper, David V.; Rutten, Victor P.M.G.; Michel, Anita Luise

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Mycobacterium bovis prevalence affects the performance of a commercial serological assay for bovine tuberculosis in African buffaloes

Van der Heijden, E.M.D.L. (Elisabeth); Cooper, David V.; Rutten, Victor P.M.G.; Michel, Anita Luise

URI: http://hdl.handle.net/2263/73818

Date: 2020-06

Abstract:

The endemic presence of bovine tuberculosis (BTB) in African buffaloes in South Africa has severe consequences for BTB control in domestic cattle, buffalo ranching and wildlife conservation, and poses a potential risk to public health. This study determined the BTB prevalence in free-ranging buffaloes in two game reserves and assessed the influence of the prevalence of mycobacterial infections on the performance of a commercial cattlespecific serological assay for BTB (TB ELISA). Buffaloes (n=997) were tested with the tuberculin skin test and TB ELISA; a subset (n=119) was tested longitudinally. Culture, PCR and sequencing were used to confirm infection with M. bovis and/or non-tuberculous mycobacteria (NTM). Prevalence of BTB, but not NTM, influenced the TB ELISA performance. Multiple testing did not increase test confidence. The findings strongly illustrate the need for development of novel assays that can supplement existing assays for a more comprehensive testing scheme for BTB in African buffaloes.

Description:

Figure S1. Examples of lesions found during the post mortem investigation in the MGR May 2016. (A) Two large (±3 × 4 cm) granulomatous, caseous tubercles in the right caudal lobe of the lungs; (B) mandibular lymph node totally full of caseous, necrotic lesions; (C) miliary tuberculosis in an African buffalo.

Figure S2. Results of the multiplex PCR on DNA from tissue isolates. (A) Results of isolates obtained from tissue samples from animals in the MGR in May 2016; (B) results of isolates obtained from tissue samples from animals in the HiP in 2016 and 2017, as well as the MGR in Jan 2017. In both gel pictures a 100bp DNA ladder was used in order to identify the amplicon size. Nuclease-free water was used as a negative control in both panels. In panel A, DNA from a known M. bovis isolate was used as a positive control, whereas sample BSL371 was used as a positive control in panel B as it had previously tested positive on this PCR. In panel B, a no template control was also included.

Figure S3. Results of the multiplex PCR on DNA from nasal swab isolates. Results of isolates obtained from nasal swabs from animals in the MGR in May 2016. A 100bp DNA ladder was used in order to identify the amplicon size. Nuclease-free water was used as a negative control. DNA from a known M. bovis isolate was used as a positive control.

Figure S4. Alignment of obtained sequences. Sequences obtained from the isolates were cleaned, trimmed and aligned using the CLC Main Workbench (Qiagen Bioinformatics, Aarhus, Denmark). The isolate number as well as the species determined by BLAST analysis are listed. Sequences are ordered according to relatedness.

Figure S5. Phylogenetic tree of the isolates obtained in the study. The sequence of M. bovis GTC 602 was included as an outgroup species, while M. asiaticum ATCC 25276 and M. moriokaense CIP 105393 were included as representatives of SGM and RGM, respectively. The tree was constructed using the neighbour-joining method with 1,000 bootstrap replicates (bootstrap values indicated at the nodes) using the CLC Main Workbench (Qiagen Bioinformatics, Aarhus, Denmark). As expected, the slow-growing mycobacteria (SGM) M. holsaticum, M. asiaticum, M. celatum, M. avium complex, M. colombiense/M. bouchedurhonense and M. vulneris/M. intracellulare [55–57] grouped with M. asiaticum ATCC 25276. Similarly, the rapid-growing mycobacteria (RGM) M. brasiliensis, M. moriokaense and M. moriokaense/M. barrassiae, M. flavescens, M. smegmatis/M. goodii, M. agri and M. rhodesiae [56–59] grouped with M. moriokaense CIP105393. Finally, a few isolates belonging to the Mycobacterium avium complex (MAC) (M. avium complex, M. colombiense/M. bouchedurhonense and M. vulneris/M. intracellulare) [60], as well as isolates belonging to the Mycobacterium moriokaense group (M. moriokaense, M. moriokaense/M. barrassiae and M. brasiliensis) [60,61], also grouped together.

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