Multilocus Sequence Typing (MLST) and Random Polymorphic DNA (RAPD) Comparisons of Geographic Isolates of Neoparamoeba perurans, the Causative Agent of Amoebic Gill Disease

Abstract

Neoparamoba perurans, is the aetiological agent of amoebic gill disease (AGD), a disease that affects farmed Atlantic salmon worldwide. Multilocus sequence typing (MLST) and Random Amplified Polymorphic DNA (RAPD) are PCR-based typing methods that allow for the highly reproducible genetic analysis of population structure within microbial species. To the best of our knowledge, this study represents the first use of these typing methods applied to N. perurans with the objective of distinguishing geographical isolates. These analyses were applied to a total of 16 isolates from Australia, Canada, Ireland, Scotland, Norway, and the USA. All the samples from Australia came from farm sites on the island state of Tasmania. Genetic polymorphism among isolates was more evident from the RAPD analysis compared to the MLST that used conserved housekeeping genes. Both techniques consistently identified that isolates of N. perurans from Tasmania, Australia were more similar to each other than to the isolates from other countries. While genetic differences were identified between geographical isolates, a BURST analysis provided no evidence of a founder genotype. This suggests that emerging outbreaks of AGD are not due to rapid translocation of this important salmonid pathogen from the same area.

Keywords: Amoebic Gill Disease; Paramoeba perurans; epidemiology; pathogen genetics.

Figure 1

Concatenated Neighbour Joining Tree based on six Multilocus Sequence Typing (MLST) gene loci (elongation factor 1 (ef1), beta tubulin (tubb), RNA polymerase large subunit 1 (Rpb1), beta actin (actb), elongation factor 2 (ef2) and succinate dehydrogenase complex flavoprotein subunit A (sdha) using average states to resolve polymorphic sites. Different groupings are represented by vertical bars. Two distinct groups can be visualized; Group 1 (Tasmania) and Group 2 (Ireland, Scotland, the USA, Canada and Norway). Branch support represents bootstrap values (1000 replications). Blue values represent the bootstrap support for the SNP duplication method (1000 replications).

Figure 2

BURST graph of all sequences used in the MLST analysis. There were five sequence types defined: 1 (Tasmanian isolates), 2 (Ireland 1), 3 (Scotland Isolates), 4 (Ireland 2) and 5 (USA, Canada and Norway). The graph shows a connection between all isolates typical of a clonal BURST configuration.

Figure 3

(A–E) Agarose gel of amplified Random Polymorphic DNA (RAPD) products for primers A1, A15, B10, B12 and B18, respectively. Lane 1, Tasmanian clone C8a; lane 2, Tasmanian clone C4a; lane 3, Tasmanian clone C4b; lane 4, Tasmanian clone C4c; lane 5, Tasmanian clone C4d; lane 6, Tasmanian isolate 1; lane 7, Tasmanian isolate 2; lane 8, Tasmanian isolate 3; lane 9, Norway; lane 10, Canada; lane 11, USA; lane 12, Ireland2; lane 13, Ireland1; lane 14, Scotland clonal isolate; lane 15, Scotland isolate 1 and lane 16, Scotland isolate 2.

Figure 4

Dendrogram created from the Jaccard coefficient distance matrix for the combined RAPD primer profiles (A1, A15, B10, B12 and B18). The colour blocks represent the geographic locations of each isolate (Australia—grey, Scotland—brown, Ireland—pink, USA—orange, Canada—purple and Norway—green).

Figure 5

(A–E) Dendrograms of the N. perurans isolates created using the neighbour joining clustering in the PyElph program for RAPD primes A1, A15, B10, B12 and B18, respectively. Colours represent geographic locations (Australia—grey, Scotland—brown, Ireland—pink, USA—orange, Canada—purple and Norway—green).