Molecular and phylogenetic analyses of Salmonella Gallinarum trace the origin and diversification of recent outbreaks of fowl typhoid in poultry farms

Abstract

Fowl typhoid (FT) and pullorum disease (PD) are two important poultry infections caused by Salmonella enterica subsp. enterica serotype Gallinarum (S. Gallinarum). S. Gallinarum strains are adapted to birds and classified into biovars Gallinarum (bvGA) and Pullorum (bvPU) as they are the causative agent of FT and PD, respectively. In Brazil, FT/PD outbreaks have been reported along the last 50 years, but there was a recent increase of FT field reports with the suspicion it could be due to virulence reversion of the attenuated live vaccine SG9R. In this study, we applied molecular biology assays and phylogenetic methods to detect and investigate S. Gallinarum isolates from commercial poultry flocks in order to understand the evolutionary history and origin of the recent FT outbreaks in Brazil. S. Gallinarum isolates were obtained from thirteen different poultry flocks with clinical signs of FT/PD from 2013 to 2015. These isolates were serotyped, tested with three specific PCR (for the detection of bvGA, bvPU and live vaccine strain SG9R) and submitted to sequencing of a variable genome region (ISR analysis). The complete genome of one bvGA strain (BR_RS12) was also compared to other S. Gallinarum complete genomes (including other two Brazilian ones: bvGA 287/91 and bvPU FCVA198). PCR detected all thirteen isolates as S. Gallinarum (eight bvGA and five bvPU), none positive for SG9R strain. ISR analysis revealed that all eight bvGA isolates showed exactly the same nucleotide sequences with 100% similarity to reference strains, while two patterns were observed for bvPU. Genome phylogeny demonstrated distinct clades for bvGA and bvPU, with the bvGA clade showing a clear subdivision including three genomes: SG9R vaccine, the respective SG9 parent strain and one SG9R revertant field isolate (MB4523). The evolutionary rate of the total S. Gallinarum genome was calculated at 6.15×10 substitutions/site/year, with 2.8 observed substitutions per year per genome (1 SNP per 4292 bases). Phylodynamics analysis estimated that at least two introductions of S. Gallinarum bvGA happened in Brazil, the first in 1885 and the second in 1950. The Brazilian bvGA genomes 287/91 and BR_RS12 analyzed here were related to the early and the late introductions, respectively. In conclusion, these results indicate the occurrence of S. Gallinarum strains associated with FT outbreaks that have been circulating for more than 50 years in Brazil and are not originated from virulence reversion of the SG9R vaccine.

Comparative genome analysis of Salmonella enterica serovar Gallinarum biovars Pullorum and Gallinarum decodes strain specific genes

“…4) of S. Gallinarum biovars. from different geographical origins has clearly divided strains into divergent clades, and Indian strain was grouped externally in the S. Gallinarum clade, as in previous study (De Carli et al 2017). Similarly, the phylogenetic relationship between S. Gallinarum biovars and S. Enteritidis has been genomically analysed, which highlighted the shared ancestry of bvP and bvG with S. Enteritidis descendent originating in a 'second' clade (Langridge et al 2015).…”

“…enterica comprises of both host-adapted and host-promiscuous pathotypes that causes a spectrum of diseases depending on the serovar or host (Jajere 2019;Cheng et al 2019). Pullorum Disease (PD) and Fowl Typhoid (FT) caused by S. Gallinarum biovars Pullorum (bvP) and Gallinarum (bvG) respectively are endemic in countries of Asia and South America leading to huge economic losses to poultry industry (Shivaprasad 2000;De Carli et al 2017;Hu et al 2019). It is interesting to note that as compared to FT, the PD reports have been low in India (Rajagopal and Mini, 2013).…”

“…Pullorum disease (PD) and fowl typhoid (FT) are two distinct septicaemic diseases caused by non-motile Salmonella enterica subsp. enterica serovar Gallinarum (S. Gallinarum) biovar Pullorum (bvP) and biovar Gallinarum (bvG), respectively, which exhibit host-speci city towards poultry and aquatic birds (De Carli et al 2017;Alves Batista et al 2018;Wilson et al 2000). The diseases caused by these invasive avian pathogens cause high morbidity and acute mortality in poultry in India and various countries of Asia, Africa and South America.…”

High-resolution diffusion pattern of human infections by Salmonella enterica serovar Napoli in Northern Italy explained through phylogeography

“…We analyzed the time and mode of evolution of the dominant clone of serovar Napoli in Northern Italy considering 2542 core SNPs of 44 isolates and we have estimated a mean evolutionary rate of 5.7x10 -7 sub/site year -1 , corresponding to a mean 2.7 substitutions/genome year -1 . This value is at the upper limit of previous estimates obtained on different Salmonella enterica serovars, varying from 0.8 to about 6.1 x10 -7 sub/site year -1 [ 25 , 26 , 29 , 43 , 44 ]. It was previously observed that human-restricted serovars (such as S .…”

“…It was previously observed that human-restricted serovars (such as S . Typhi and Paratyphi A) have evolutionary rates on average lower (0.8–2 x10 -7 sub/site year -1 ) than those estimated for less restricted serovars such as Kentuky, Agona, Gallinarum (3.95-6x10 -7 -sub/site year -1 ) [ 25 , 26 , 29 , 43 , 44 ].…”