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A msp1alpha polymerase chain reaction assay for specific detection and differentiation of Anaplasma marginale
A E Lew1, R E Bock, C M Minchin
1Queensland Department of Primary Industries, Agency for Food and Fibre Sciences, c/o Animal Research Institute, Locked Mail Bag No. 4, Qld 4105, Moorooka, Australia. ala.lew@dpi.qld.gov.au
Abstract:
Anaplasma marginale is the causative agent of bovine anaplasmosis, a disease which can be protected by vaccination with the less pathogenic Anaplasma species, A. centrale. Currently, there is no polymerase chain reaction (PCR) assay available which differentiates between different species of Anaplasma or which can differentiate isolates of A. marginale within outbreaks and between different countries. A molecular test specific for A. marginale would be ideal for the identification of Anaplasma species in wild ruminants, as possible reservoirs of anaplasmosis, and to differentiate between A. marginale from A. centrale. A PCR assay was designed to amplify the major surface protein 1alpha gene of the rickettsial bovine pathogen, A. marginale both as an inter- and intra-specific test. The test did not amplify A. centrale or A. ovis, and discriminated A. marginale by amplifying repeat regions within the msp1alpha gene which vary in number between many isolates. The nested A. marginale amplicons varied in size from 630 to 1190bp representing one to eight internal repeats. All 22 Australian isolates tested amplified a 630bp product (one repeat) in contrast to all 19 non-Australian isolates tested. Eight sequences from Australian isolates from different geographical regions confirmed the conserved nature of the Australian A. marginale msp1alpha genes. The Australian 'repeat unit' MSP1a deduced amino acid sequence has been designated as Australian type 1. The msp1alpha PCR method developed here enabled the amplification and comparison of A. marginale isolates originating from North and South America, Africa, Israel and Australia. The method is sensitive and specific for A. marginale. Although additional msp1alpha products were amplified from at least two Australian isolates, the results suggest limited introduction of A. marginale into Australia.
Insights
A new polymerase chain reaction (PCR) assay accurately identifies Anaplasma marginale, the cause of bovine anaplasmosis. This molecular test differentiates A. marginale from other species and distinguishes between global isolates, aiding disease control and research.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Parasitology
Background:
- Anaplasma marginale causes bovine anaplasmosis, a significant disease in cattle.
- Current diagnostic methods lack specificity to differentiate Anaplasma species or strains.
- A specific molecular test is needed for accurate identification and epidemiological studies.
Purpose of the Study:
- To develop a species-specific polymerase chain reaction (PCR) assay for Anaplasma marginale.
- To differentiate Anaplasma marginale from other Anaplasma species, including A. centrale and A. ovis.
- To distinguish between different isolates of Anaplasma marginale based on genetic variation.
Main Methods:
- A nested PCR assay was designed to amplify the major surface protein 1alpha (msp1alpha) gene of Anaplasma marginale.
- The assay targeted repeat regions within the msp1alpha gene, which vary in number among isolates.
- The PCR test was validated using isolates from Australia and other global regions.
Main Results:
- The developed PCR assay specifically amplified Anaplasma marginale, showing no amplification with A. centrale or A. ovis.
- The assay differentiated between Australian and non-Australian Anaplasma marginale isolates based on the number of msp1alpha repeats.
- Australian isolates predominantly exhibited a single repeat (630bp), while non-Australian isolates showed variation, indicating genetic diversity.
Conclusions:
- A sensitive and specific PCR assay targeting the msp1alpha gene has been developed for Anaplasma marginale.
- This molecular tool enables accurate differentiation of Anaplasma species and A. marginale isolates globally.
- The findings provide a valuable resource for epidemiological surveillance and control of bovine anaplasmosis.
