Horizontal gene transfer of the pirAB genes responsible for Acute Hepatopancreatic Necrosis Disease (AHPND) turns a non-Vibrio strain into an AHPND-positive pathogen

In the past decade, shrimp farms, particularly those established in Asia, Mexico and South America suffered from the outbreak of an emergent penaeid shrimp disease known as Acute Hepatopancreatic Necrosis Disease (AHNPD). The PirA and PirB toxins produced by plasmid pVA1 in Vibrio parahaemolyticus were reported to cause the AHPND pathology. More recent research demonstrated that V. parahaemolyticus is not the only species that can cause AHPND, as other Vibrio species were also found to contain PirAB-containing plasmid. The present study assessed the Horizontal Gene Transfer (HGT) of AHPND that transforms genes (pirA and pirB) from AHPND positive V. parahaemolyticus to non-AHPND and non-vibrio species identified as Algoriphagus sp. strain NBP. The HGT of pirA and pirB genes from the AHPND positive V. parahaemolyticus to Algoriphagus sp. strain NBP was found to occur at different temperatures. The conjugation efficiency rate (n°) of pirAB from V. parahaemolyticus to Algoriphagus sp. strain NBP at 30°C and 40°C showed 80-91% efficiency. Shrimp challenged with the pirA and pirB positive Algoriphagus sp. strain NBP also demonstrated typical pathognomonic AHPND lesions during the histopathologic examination. Author summary AHPND is a significant threat to the shrimp industry leading to high losses. The results demonstrated that the conjugative transfer of the pirA and pirB positive V. parahaemolyticus (donor strain) to a non-Vibrio and non-pathogenic bacterium (recipient strain), successfully transformed the non-pathogenic bacterium into a disease-causing strain with a disease-causing capability similar to the donor strain. Initially, V. parahaemolyticus that express the PirA and PirB toxins which encoded by a conjugative plasmid cause sloughing and degeneration of shrimp hepatopancreatic.


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It has been a decade since the emergence of Acute Hepatopancreatic Necrosis Disease 68 (AHPND) that has caused global losses of more than $ 1 billion per year, and mortality rates 69 up to 100% in the shrimp farming industries within the regions of Asia, Mexico, South America 70 and Texas [1][2]. The etiological agent has been initially identified as Vibrio parahaemolyticus 71 carrying a plasmid containing toxin genes (pirA and pirB). The two toxin subunits, PirA and AHPND-like histopathology [3,5,7 ]. Thus far, all pirA and pirB positive strains which causes 86 AHPND belong to species within the Harveyi clade of vibrios (i.e. a clade of species closely 87 related to V. harveyi) [3][4][5][6][7]. However, very little is known on the possibility of conjugative 88 transfer of these genes to non-Vibrio spp. Such a gene transfer would have far-reaching 89 implications for disease control as it would be able to also turn non-Vibrio strains into AHPND-5 90 causing agents. In order to investigate potential horizontal gene transfer of the pirA and pirB 91 genes from V. parahaemolyticus to a non-AHPND and non-Vibrio bacterium, we assessed 92 transfer of the genes to an Algoriphagus sp. strain NBP isolated from marine microalgae 93 (Nannochloropsis sp.).

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Isolation and of a pirAB negative non-Vibrio strain from a microalgal culture 97 Eight strains were isolated from the microalgal culture. An isolate with a pink colony and 98 creamy texture on MA plates, denoted NBP, was selected for further work as this colony 99 morphology enabled us to easily differentiate the isolate from Vibrio parahaemolyticus 100 BpShHep31 (Fig 1). The selected isolate was confirmed to be negative for the pirA and pirB 101 genes using PCR with specific primers (VpPirA-284F, VpPirA-284R, VpPirB-392F, and 102 VpPirB-392R).

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Identification of the pirAB negative isolate 105 A BLAST search revealed that the 16S rDNA gene sequence of isolate NBP showed 99% 106 similarity to that of A. marincola strain SW-2 (GenBank accession MK583623). The 16S rDNA 107 of the isolate formed a monophyletic taxon with A. marincola strain SW-2 with a posterior 108 probability (PP = 0.67) (Fig 2). Hence, the isolate is further denoted as Algoriphagus sp. strain  Co-culture of isolate NBP and V. parahaemolyticus BpShHep31 and screening for the 113 presence of pirAB genes in colonies re-isolated after co-culture 114 The results showed that although Algoriphagus sp. strain NBP was negative for pirA and pirB 115 prior to co-culture, several colonies of the isolate that were picked up from MA plates after co-  The density of Algoriphagus sp. strain NBP in the cocultures significantly increased (P < 0.05) 122 at higher temperature (Fig 4) We calculated the conjugation efficiency of pirAB genes and found that it was high (80-81%) 131 at 30°C and 40°C (Table 1). Furthermore, no significant differences (P > 0.05) were observed 132 between the conjugation efficiency of pirAB at 30-40°C.  The presence of pirA and pirB genes was determined using PCR with specific primers.

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Genomic DNA was extracted from grown cultures using the Geneaid kit (Taiwan) and was 242 purified by adhering to the protocol provided by the manufacturer. The primers used for PCR 243 amplification of the pir genes are listed in   MAFFT (v7.365) [23], and the aligned sequences were manually corrected via BioEdit.

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Characters that were aligned ambiguously had been excluded from the analysis. Roseivirgo  The Algoriphagus sp. NBP colonies that were picked after plating of the coculture with V. 295 parahaemolyticus strain BpShHep31 were verified to be free from contamination by V. 296 parahaemolyticus by performing PCR with specific primers for the V. parahaemolyticus toxR 297 gene (which is absent in Algoriphagus) ( Table 2). Sterile 10 µL pipette tip was used to pick the 298 colonies to avoid any contamination. The DNA of the colonies were extracted by adding 10 µL 299 of sterile distilled water followed by heating at 94°C for 5 minutes. Then, the samples were 300 cooled at 4°C and centrifuged at 10,000 rpm for 5 minutes. The supernatant of the samples was 301 transferred to another sterile 0.2 mL tube. The Algoriphagus sp. strain NBP colonies were 302 screened for the presence of the pirAB genes using the VpPirA and VpPirB primers ( Table 2). 303 We then calculated the conjugation efficiency (%) as follows: