Isolation of Mycobacterium avium subspecies paratuberculosis from Ugandan cattle and strain differentiation using optimised DNA typing techniques

doi:10.1186/1746-6148-8-99

BMC Vet Res. 2012; 8: 99.

Published online 2012 June 29. doi: 10.1186/1746-6148-8-99

PMCID: PMC3416654

Isolation of Mycobacterium avium subspecies paratuberculosis from Ugandan cattle and strain differentiation using optimised DNA typing techniques

Julius Boniface Okuni,¹ Chrysostomos I Dovas,² Panayiotis Loukopoulos,² Ilias G Bouzalas,² David Patrick Kateete,³ Moses L Joloba,³ and Lonzy Ojok¹

¹College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, P.O.Box 7062, Kampala, Uganda

²Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, 54124, Greece

³Department of Medical Microbiology, College of Health Sciences, Makerere University, P.O.Box 7062, Kampala, Uganda

Corresponding author.

Julius Boniface Okuni: jbok/at/vetmed.mak.ac.ug; Chrysostomos I Dovas: dovas/at/vet.auth.gr; Panayiotis Loukopoulos: plouk/at/vet.auth.gr; Ilias G Bouzalas: ilbouzal/at/vet.auth.gr; David Patrick Kateete: davidkateete/at/gmail.com; Moses L Joloba: moses.joloba/at/case.edu; Lonzy Ojok: lonzy/at/vetmed.mak.ac.ug

Received February 24, 2012; Accepted June 18, 2012.

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Abstract

Background

The occurrence of paratuberculosis in Ugandan cattle has recently been reported but there is no information on the strains of Mycobacterium avium subspecies paratuberculosis (MAP) responsible for the disease. The aim of this study was to isolate and characterise MAP from seropositive cattle and paratuberculosis lesions in tissues obtained from slaughtered cattle in Uganda.

Results

Twenty one isolates of MAP were differentiated into 11 genotype profiles using seven genotyping loci consisting of Insertion Sequence 1311(IS1311), Mycobacterial interspersed repeat units (MIRU) (loci 2, 3), Variable number tandem repeats (VNTR) locus 32 and Short sequence repeats (SSR) (loci 1, 2 and 8). Three different IS1311 types and three MIRU 2 profiles (7, 9, 15 repeats) were observed. Two allelic variants were found based on MIRU 3 (1, 5 repeats), while VNTR 32 showed no polymorphism in any of the isolates from which it was successfully amplified. SSR Locus 1 revealed 6 and 7

G1 repeats among the isolates whereas SSR locus 2 revealed 10, 11 and 12

G2 repeats. SSR locus 8 was the most polymorphic locus. Phylogenetic analysis of SSR locus 8 sequences based on their single nucleotide polymorphisms separated the isolates into 8 genotypes. We found that the use of Ethylene glycol as a PCR additive improved the efficiency of the PCR reactions for MIRUs (2, 3), VNTR 32 and SSR (loci 1 and 2).

Conclusions

There is a high strain diversity of MAP in Uganda since 21 isolates could be classified into 11 genotypes. The combination of the seven loci used in this study results into a very precise discrimination of isolates. However analysis of SNPs on locus alone 8 is very close to this combination. Most of the genotypes in this study are novel since they differed in one or more loci from other isolates of cattle origin in different studies. The large number of MAP strains within a relatively small area of the country implies that the epidemiology of paratuberculosis in Uganda may be complicated and needs further investigation. Finally, the use of Ethylene glycol as a PCR additive increases the efficiency of PCR amplification of difficult templates.

Keywords: Mycobacterium avium subspecies paratuberculosis, Cattle, Uganda, SSR, MIRUs, Genotyping, IS1311 PCR-REA

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