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Isolation of Mycobacterium avium subspecies paratuberculosis from Ugandan cattle and strain differentiation using

Julius Boniface Okuni¹, Chrysostomos I Dovas, Panayiotis Loukopoulos

¹College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, P.O. Box 7062, Kampala, Uganda. jbok@vetmed.mak.ac.ug

BMC Veterinary Research

|July 4, 2012

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.

Insights

This study reveals high Mycobacterium avium subspecies paratuberculosis (MAP) strain diversity in Uganda, with 21 isolates showing 11 distinct genotypes. Further investigation is needed to understand the complex epidemiology of paratuberculosis in the region.

Area of Science:

Veterinary Microbiology
Bovine Diseases
Molecular Epidemiology

Background:

Paratuberculosis has been reported in Ugandan cattle, but the specific strains of Mycobacterium avium subspecies paratuberculosis (MAP) causing the disease remain uncharacterized.
Limited information exists regarding the genetic diversity of MAP circulating within the Ugandan cattle population.

Purpose of the Study:

To isolate and genetically characterize Mycobacterium avium subspecies paratuberculosis (MAP) from cattle in Uganda.
To determine the strain diversity of MAP responsible for paratuberculosis in Ugandan cattle populations.

Main Methods:

Isolation and genotyping of MAP from seropositive cattle and tissue lesions.
Utilized seven genotyping loci: IS1311, MIRU (2, 3), VNTR 32, and SSR (1, 2, 8).
Optimized PCR amplification using Ethylene glycol as an additive for challenging templates.

Main Results:

Twenty-one MAP isolates were classified into 11 distinct genotype profiles.
High polymorphism was observed across several loci, particularly SSR locus 8, which separated isolates into 8 genotypes.
The use of Ethylene glycol significantly improved PCR efficiency for multiple loci.

Conclusions:

Significant MAP strain diversity exists in Uganda, with 11 genotypes identified from 21 isolates.
The combined genotyping loci provide precise discrimination, with SSR locus 8 analysis showing similar discriminatory power.
Novel MAP genotypes were identified, suggesting a complex and understudied paratuberculosis epidemiology in Uganda.

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