Multiplex PCR protocol developed to typify existing sub-lineages in Chile of the BKD agent

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By Milthon Lujan

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A collaborative research study between research teams from the Pontificia Universidad Católica de Chile (PUC), the Andrés Bello University (UNAB), and the INCAR Center, allowed the development of a new multiplex PCR protocol to typify the existing sub-lineages in Chile of the BKD agent, Renibacterium salmoninarum.

Renibacterium salmoninarum (Rs) is the etiologic agent of bacterial kidney disease (BKD), which significantly affects farmed and wild salmonids worldwide.

The research whose authors are Patricia Mora-Salas, Sebastián Zapararte, Pamela Villouta, Henry Araya-León, Ruben Avendaño-Herrera, Francisco Melo, Fernando O. Mardones, who created a novel multiplex in silico PCR protocol, and validated it at based on laboratory work with 27 Chilean R. salmoninarum isolates recovered from kidney, heart, liver and/or spleen samples of Atlantic salmon and Coho salmon farmed from the Araucanía Region to the Magallanes Region during outbreaks that occurred between 2014 and 2021.

The experts qualified this multiplex PCR-Sanger sequencing approach “as a valuable tool for the rapid and reliable typing of each of the four sub-lineages described for Chilean R. salmoninarum isolates.” Furthermore, the investigators highlighted that “the sublineage typing method is lower cost and requires less work and effort compared to the task of sequencing the whole genome of a sample and will facilitate future retrospective or prospective epidemiological studies involving infections by R. salmoninarum in Chilean fish farms and fattening centers”.

Although the complete genome of R. salmoninarum (~3.1 million nucleotides) is highly conserved, previous genomic epidemiology studies identified two lineages (1 and 2) and within lineage 1 four sub-lineages within which the Chilean isolates: 1a, 1b, 1c, and 1d. In the study, researchers from PUCV, UNAB, and INCAR identified some nucleotide sequences that presented differences in very specific regions of the genome, commonly called single nucleotide polymorphisms (SNPs).

Thus, the scientists discovered a set of three nucleotides (SNPs 2088, 2092, and 2123) positioned within the genome in a way that allowed their simultaneous amplification by PCR, and which, in turn, were unique for each sub-lineage, allowing their differentiation.

Within the practical applications of these results, the Principal Investigator of the INCAR Center and UNAB academic, Dr. Ruben Avendaño, explained that knowing the sub-lineage of R. salmoninarum endemic to a fish farm would allow monitoring if it occurs. outbreaks after the transfer of salmon to fattening at sea, and even determine if infected salmon could have bacteria from two or more sub-lineages in the same center or outbreak.

“The protocol is validated and the applications are very valuable. For example, the diagnostic laboratories of the network could implement it for the characterization of the lineages, associating them with the different fish farms or fattening centers. Even an isolate that we have categorized as highly virulent presents a specific lineage, therefore, we will carry out more studies again in collaboration to associate the pathogenic potential of R. salmoninarum and its sub-lineages”, explained Dr. Avendaño-Herrera.

Renibacterium salmoninarum is the etiologic agent of bacterial kidney disease (BKD), which significantly affects farmed and wild salmonids worldwide. In Chile, the productive impact of BKD is not quantified, but losses due to mortality in freshwater are reflected in growth, affecting an estimate of up to approximately 20% of the population, depending on the species.

Mora-Salas, P., Zapararte, S., Villouta, P., Araya-León, H., Avendaño-Herrera, R., Melo, F., & Mardones, F. O. (2023). Method for lineage typing of epidemic Renibacterium salmoninarum in Chilean salmon farms. Journal of Fish Diseases, 00, 1– 8. https://doi.org/10.1111/jfd.13761

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