Evaluation of the discriminatory power of spoligotyping and 19-locus mycobacterial interspersed repetitive unit-variable number of tandem repeat analysis (MIRU-VNTR) of Mycobacterium bovis strains isolated from cattle in Algeria

Bovine tuberculosis (bTB) caused by Mycobacterium (M.) bovis and M. caprae is a transmissible disease of livestock, notifiable to the World Organization for Animal Health (OIE). BTB particularly affects cattle and small ruminants and can be transmitted to humans thereby posing a significant threat to veterinary and public health worldwide. M. bovis is the principal cause of bTB in Algeria. In order to better understand the route of spreading and elaborate an eradication program, isolation and characterization of mycobacteria from Algerian cattle was performed. Sixty strains belonging to the M. tuberculosis complex were analyzed by spoligotyping, thereof 42 by 19-locus-MIRU-VNTR-typing. Spoligotyping revealed 16 distinguishable patterns (Hunter-Gaston discriminatory index [HGDI] of 0.8294), with types SB0120 (n = 20) and SB0121 (n = 13) being the most frequent patterns, representing 55% of the strains. Analyses based on 19-locus-MIRU-VNTR yielded 32 different profiles, five clusters and one orphan pattern, showing higher discriminatory power (HGDI = 0.9779) than spoligotyping. Seven VNTR-loci [VNTR 577 (alias ETR C), 2163b (QU11b), 2165 (ETR A), 2461 (ETR B), 3007 (MIRU 27), 2163a (QUB11a) and 3232 (QUB 3232)] were the most discriminative loci (HGDI ˃ 0.50). In conclusion, 19-locus-MIRU-VNTR yielded more information than spoligotyping concerning molecular differentiation of strains and better supports the elucidation of transmission routes of M. bovis between Algerian cattle herds.

The authors have declared that no competing interests exist.

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Bovine tuberculosis (bTB) represents a veterinary and public health threat of global relevance.

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Despite the importance of the disease, there is no official data concerning the true prevalence 52 of bTB in many low and middle-income countries. This also holds for Algeria, despite the fact 53 that bTB is a notifiable animal disease [1]. Although multiple disease control plans are  In Algeria, we suspect that most bTB-infected herds are geographically clustered in the north- Petroff's method [13,14]. Briefly, an amount of 5 ml NaOH solution (4%) was added to about 98 10 g of tissue, minced by a mortar in 4 ml of sterile distilled water, and incubated for 15 min.

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The suspension was diluted by 10 ml of sterile distilled water and then centrifuged at 3000 x g 100 for 20 min. The pellet was used as an inoculum to solid Löwenstein-Jensen medium.   The predominant spoligotypes found were SB0120 (n = 20) and SB0121 (n = 13), both 167 belonging to the family BOV_1 and differing in only one spacer (no. 21), followed by SB0134 168 (n = 7), SB2521 (n = 5), and SB0828 (n = 2). The currently most prevalent spoligotypes 169 SB0120, SB0121, and SB0134 were already detected by a study conducted 10 years earlier in 170 Algeria [23]. Of note, the two new types SB2522 and SB2523 as well as three additional 171 spoligotypes, namely SB0119 (n = 1), SB0848 (n = 1), and SB1542 (n = 2) have not been 172 reported in Algeria before.    205 Out of 60 strains of this study, VNTR profiles could be generated for 42 M. bovis strains.

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Missing success in the remaining cases might have been due to low DNA quality or quantity.

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A panel of 19 loci was chosen for conducting the MIRU-VNTR (Table 2).

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Overall, seven loci (VNTR577, 2163a, 2163b, 2165, 2461, 3007, and 3232) presented a high 222 discriminatory power (HGDI ˃ 0.50, Table 2). Our findings corroborate therefore earlier data 223 obtained in Tunisia [25] which showed a high resolution of VNTR2163a, 2163b, 2461, 2165, 224 and 3232, while the four loci VNTR577, 2165, 2461, and 3007, showed a high discriminatory 225 power for Algerian strains also in previous data sets [42]. Furthermore, this is consistent with  (Table   244 3).  The discriminatory capacity of spoligotyping is limited since diversity is measured at a single 313 locus prone to convergent evolution and phylogenetic distances cannot be reliably inferred [55]. cluster is divided into two groups: the BCG-like group, represented by SB0121, and the ancestor 318 BCG-like SB0120. In the present study, all M. bovis strains lacked spacers 3, 9, 16, and 39 to 319 43 and were therefore grouped to the BCG cluster (Table 1). On the African continent, the 320 ancestral BCG-like cluster is predominantly found in Algeria, Zambia [56], and Mozambique [57]. The cluster Af1, Af2, and Eu1 have not been found in cattle outside of the previous 322 mentioned regions [24]. Further studies, implicating WGS technology, will be required to