pirABvp-Bearing Vibrio parahaemolyticus and Vibrio campbellii Pathogens Isolated from the Same AHPND-Affected Pond Possess Highly Similar Pathogenic Plasmids

Acute hepatopancreatic necrosis disease (AHPND) is a severe shrimp disease originally shown to be caused by virulent strains of Vibrio parahaemolyticus (VPAHPND). Rare cases of AHPND caused by Vibrio species other than V. parahaemolyticus were reported. We compared an AHPND-causing V. campbellii (VCAHPND) and a VPAHPND isolate from the same AHPND-affected pond. Both strains are positive for the virulence genes pirABvp. Immersion challenge test with Litopenaeus vannamei indicated the two strains possessed similar pathogenicity. Complete genome comparison showed that the pirABvp-bearing plasmids in the two strains were highly homologous, and they both shared high homologies with plasmid pVA1, the reported pirABvp-bearing plasmid. Conjugation and DNA-uptake genes were found on the pVA1-type plasmids and the host chromosomes, respectively, which may facilitate the dissemination of pirABvp. Novel variations likely driven by ISVal1 in the genetic contexts of the pirABvp genes were found in the two strains. Moreover, the VCAHPND isolate additionally contains multiple antibiotic resistance genes, which may bring difficulties to control its future outbreak. The dissemination of the pirABvp in non-parahaemolyticus Vibrio also rises the concern of missing detection in industrial settings since the isolation method currently used mainly targeting V. parahaemolyticus. This study provides timely information for better understanding of the causes of AHPND and molecular epidemiology of pirABvp and also appeals for precautions to encounter the dissemination of the hazardous genes.

V. parahaemolyticus is a Gram-negative bacterium that widely inhabits the marine and estuarine environments and it can cause disease in human and animals (Makino et al., 2003;Thompson et al., 2004). A recent study has demonstrated that VP AHPND harbors a plasmid that expresses a deadly toxin Pir vp (constituted by PirA vp and PirB vp ), which is homologous to the Photorhabdus insect-related (Pir) binary toxin (Lee et al., 2015). The Pir vp toxin was firstly characterized in the VP AHPND strain 3HP and was produced by plasmid pVA1 (Lee et al., 2015). The Pir vp toxin is encoded by the pirA vp and pirB vp genes that are located within a fragment delimited by two identical inversely-oriented insertion sequences (IS) ISVal1. It is believed the two ISVal1 with the region in between form a composite transposon called Tn6264 (Han et al., 2017) or pirAB-Tn903 (Xiao et al., 2017). Interestingly, missing of the fragment was seen on a plasmid sharing homology with pVA1 from a non-AHPND-causing strain M2-36, suggesting that a natural deletion or insertion of pirAB vp might have occurred (Lee et al., 2015). Other variants with partial deletion were also found (Han et al., 2017). The plasmid pVA1 also carries a cluster of genes related to conjugative transfer (Lee et al., 2015); hence, this plasmid may potentially be able to transfer not only among V. parahaemolyticus strains but also to different bacterial species. Indeed, the existence of pirAB vp has been reported in harveyi-like Vibrio (one isolate from Vietnam) (Kondo et al., 2015), V. owensii (one isolate from China) (Liu et al., 2015;Xiao et al., 2017) and V. campbellii (one isolate from China and four from Latin America) (Dong et al., 2017a;Han et al., 2017). We have previously reported the first AHPND-causing V. campbellii (VC AHPND ) strain, 20130629003S01, isolated from Guangxi, China, which contains the pirAB vp and showed pathogenicity in shrimp (Dong et al., 2017a).
In order to better understand of the spread of pirAB vp , in this study, we compared the pathogenicity and genomic features of the previously reported VC AHPND with that of an isolate of VP AHPND from the same batch of shrimp with AHPND in the same pond. Both strains were pirAB vp -positive and had a similar pathogenic capacity. We further compared their complete genomes and found that the sequences of the pirAB vp -bearing plasmids of both strains were highly homologous, and they shared high homologies with the sequence of pVA1. It suggests that plasmid-mediated interspecies transfer of the hazard genes might have occurred.

Sample Collection, Bacteria Isolation and Identification
In June of 2013, samples were collected from AHPND-suspected shrimp farms in Guangxi, China. Hepatopancreas (HP) from diseased shrimp were aseptically disaggregated and streaked on thiosulfate citrate bile salts sucrose (TCBS) plates at 28 • C for 12 h. After pure cultures were obtained, a partial 16S rRNA region was amplified with primers 16S_27F and 16S_1492R (Lane, 1991) (Table S1) and sequenced. Partial rpoD, rctB, and toxR genes were amplified and sequenced as described by Pascual et al. (2010) (Table S1). The concatenated sequences of 16S rRNA, rpoD, rctB, and toxR loci were aligned. Then the phylogenetic tree was constructed using neighbor-joining analysis with maximum composite likelihood model in MEGA 5 (Tempe, AZ, USA) with 1,000 bootstrap replications. The nucleotide sequences from strain Vp 2S01 have been submitted to the GenBank database under accession numbers MF621565 (16S rRNA), MF621566 (toxR), MF621567 (rpoD) and MF621568 (rctB). 16S rRNA, rpoD, rctB and toxR allele sequences of Vc 3S01 were described by our previously report (Dong et al., 2017a). All 16S rRNA, rpoD, rctB, and toxR allele sequences of reference strains were described by Pascual et al. (2010).
Detection of pirA vp and pirB vp in the VP AHPND Isolate Bacterial isolates Vp 2S01 and Vc 3S01 were cultured overnight in Tryptic soy broth with 2% NaCl (TSB+) at 28 • C. One milliliter (mL) of the broth culture was boiled for 10 min at 95 • C, and the supernatant was obtained by centrifugation, diluted 10-fold with distilled water and used as the template for PCR. PCR was performed using primers VpPirA and VpPirB (Han et al., 2015) (Table S1) as previously described. Protein products were examined by SDS-PAGE and mass spectrometry as previously described (Laemmli, 1970;Wang et al., 2011).

Ethics Statement
Since the Ethical Principles and Guidelines for the Use of Animals of the National Research Council of China applies to vertebrates only, there is no official standard for invertebrates, we adapted its principles to shrimp.

Litopenaeus vannamei Challenge Test
Before the challenge test, ∼1 g healthy white shrimp (L. vannamei) were acclimated in the laboratory for 3 days in 50 L seawater at salinity 30 with constant aeration in plastic tanks (density 20 shrimp/tank) at 27 ± 2 • C. For the immersion challenge, 20130629002S01 and 20130629003S01 were cultured in TSB+ at 28 • C until the OD 600 reached 0.8-0.9 (approximately 8-12 h). Immersion challenge was performed following the immersion bioassay protocol described by Tran et al. (2013). Simply, the bacterial suspension was adjusted to 1 × 10 8 cfu·mL −1 (with TSB+), and 20 shrimp in each group were immersed in 4 L of this suspension for 15 min. The shrimp and bacterial suspension were then poured into tanks containing 50 L of seawater to give a final bacterial density of 1 × 10 6 cfu·mL −1 . The control shrimp were immersed in TSB+ medium. The mortality of each group was recorded every 6 h. Moribund shrimp were fixed with Davidson's alcohol-formalin-acetic acid (DAFA) fixative for histopathological examination. All experiments were done in triplicate.

Histological Confirmation of AHPND
All shrimp sampled for histopathology purposes were fixed with DAFA for 24 h and stained with hematoxylin and eosin (H&E) using routine histological methods described by Lightner (1996). The histological sections were analyzed and photographed by a light microscopy system.

Sequencing, Assembly and Annotation of the Genome of the VP AHPND Isolate
Genomic DNA isolation, sequencing, assembly and annotation were performed as previously described (Dong et al., 2017b). Antibiotic resistance genes were searched against the ResFinder (Zankari et al., 2012). DNA-uptake genes were identified by Blast against the counterparts reported in Vibrio cholera (Seitz and Blokesch, 2013).

Statistics
All statistical analyses were performed with SPSS, version 17.0 (SPSS Inc., Chicago, IL, USA). Cumulative mortality was compared by using One-Way ANOVA test. A P-value less than 0.05 was considered statistically significant.

Nucleotide Sequence Accession Numbers
Complete genome sequences of 20130629002S01 (V. parahaemolyticus) have been deposited in GenBank under the accession CP020034-CP020037.

Two Distinct pirAB vp -Positive Strains Isolated from an AHPND Epidemic Pond Displayed Similar Pathogenicity
In June of 2013, an AHPND-suspected outbreak occurred in a shrimp farm in Guangxi. We have previously reported an AHPND-causing isolate 20130629003S01 (3S01 for short) from the farm, which has been identified as a pirAB vp -positive V. campbellii (Dong et al., 2017a). In addition, we isolated another strain, 20130629002S01 (2S01 for short) from the same batch of diseased Litopenaeus vannamei in a same pond of the farm. We found strain 2S01 was a pirAB vp -positive V. parahaemolyticus (Figure 1). Thus, for short, we used "Vp 2S01" for strain 2S01 and "Vc 3S01" for strain 3S01 in this paper.
Shrimp immersed with Vp 2S01 or Vc 3S01 suspension were observed to develop typical gross signs of AHPND within 6 h. AHPND shrimp from the Vp 2S01-and Vc 3S01-infected groups had a pale, atrophied HP, and an empty stomach (ST) and midgut (MG), compared to the normal ones that had a normal size HP with dark orange color and a full ST and midgut MG. The survival pattern of different groups was illustrated in Figure 2A. The control group showed no mortality whereas Vp 2S01-and Vc 3S01-infected groups showed 100% mortality within 24 h. As shown in Figure 2B, Vp 2S01 and Vc 3S01 had similar pathogenicity (P > 0.05). Histopathological examination of moribund shrimp samples from Vp 2S01-and Vc 3S01infected shrimp were used to confirm AHPND, which also revealed similar presence of AHPND lesions in HP ( Figure S1).

Genomic Feature Comparison of Vp 2S01 and Vc 3S01
The genome of Vc 3S01 has been previously sequenced (Dong et al., 2017b). Genome sequences of Vp 2S01 were determined here using the PacBio RS II sequencing platforms with 189× coverage. Vp 2S01 contains two circular chromosomes (I and II) and two circular plasmids (pVPGX1 and pVPGX2), while Vc 3S01 contains two circular chromosomes (I and II) and four plasmids (pVCGX1 to pVCGX4) (Dong et al., 2017b). Plasmids pVPGX1 and pVCGX1 are homologous to V. paraheamolyticus plasmids pVA1, pVPA3-1 with overall 99% nucleotide identities and partial pFORC4 (accession no. CP009849) with a 94% identity.
Significantly, genes related to DNA transfer were found in both strains. Conjugative transfer were identified on plasmids pVPGX1, pVCGX1, pVCGX3 and pVCGX4 and the chromosome I of Vc 3S01. Plasmids pVPGX1 and pVCGX1 carry tra-trb genes that are highly similar to the counterparts on pVA1 (Lee et al., 2015). The plasmids pVCGX3, pVCGX4 and the chromosome I of Vc 3S01 contain virB/D clusters (Table S2). The ones on pVCGX3 and the chromosome I are near identical, but distinct to the one on pVCGX4. The virB/D genes encoding a type IV secretion system were well known on the Agrobacterium tumefaciens plasmid Ti as being responsible for effector translocation and DNA conjugation (Shirasu et al., 1990;Christie, 2004). In addition, many other homologous virB/D clusters were characterized to be self-transmissible modules of conjugative plasmids and integrative and conjugative elements (ICEs) (Bi et al., 2013). Moreover, intact sets of genes related to DNA uptake were identified on the chromosomes I of both strains ( Table 1). Their products display 35.7-95.4% amino acid identities to the counterparts of V. cholerae O1 biovar El Tor str. N16961, which are also encoded by chromosome I. The DNA-uptake genes in V. cholerae N16961 has been proved to be required for efficient natural transformation (Seitz and Blokesch, 2013).
Antibiotic resistance genes were found in both strains, which will be described later. Neither Vp 2S01 nor Vc 3S01 contain genes (scrB) that encode the sucrose hydrolase. Both Vp 2S01 and Vc 3S01 formed green and round colonies on TCBS plate. However, genes encoding the sucrose hydrolase were found in the genome sequence of AHPND-causing strain SH14 of V. owensii (Liu et al., 2015).

Complete Plasmid Sequences Comparison of pirAB vp -Bearing Plasmids
The pirAB vp genes located on plasmids pVPGX1 in Vp 2S01 and pVCGX1 in Vc 3S01. The pVPGX1 and pVCGX1 displayed a 99% nucleotide identity spanning the entire sequences, while each of the two plasmids displayed a 96-100% nucleotide identity to pVA1 (Figure 4A). Further comparative analysis found that the two plasmids shared high homologies with other pirAB vp -bearing plasmids from AHPND-causing Vibrio strains ( Figure S2). Importantly, pVPGX1 and pVCGX1 shared the closest relationship among all the compared plasmids from AHPND-causing Vibrio (Figure 3). More interestingly, different versions of the genetic contexts of pirAB vp (pirAB vp contexts) were observed in all plasmids when compared ( Figure S2).

Sequence Analysis of the pirAB vp Contexts of Plasmids pVPGX1 and pVCGX1
In pVA1, the pirAB vp genes locate within a 5.5-kb fragment ended with two inversely oriented copies of ISVal1 which belonged to the IS903 group of the IS5 family (length includes the two IS elements). The pirAB vp were located among some small ORFs between the two ISVal1. ISVal1 was 1,054 bp in length and demarcated by 18-bp perfect inverted repeats (5 ′ -GGCTTTGTTGCGTAATTC-3 ′ ). It displayed a 92% nucleotide identity to ISVa2, which was initially found in V. anguillarum (Tolmasky and Crosa, 1995). In pVPGX1, an inversion of the 5.5-kb pirAB vp fragment was seen. Transformation of two configurations was likely due to recombination between two opposite ISVal1 (Partridge, 2011). In contrast, the pirAB vp contexts in pVCGX1 had a more complex structure. The pirAB vp fragment had an extra copy of ISVal1, which divided the fragment into two parts. The left part was inversely syntenic to pVA1, while the right part was directly syntenic to pVA1 (Figure 4B). In addition, an ISVal1 located downstream of trbN and a 217bp fragment within the trb gene cluster in pVA1 were absent in pVPGX1 and pVCGX1 ( Figure 4A).
We also analyzed the pirAB vp contexts in 12 other sequenced AHPND-causing Vibrio genomes (Table S3). Novel variant was not detected using the current data except that the pirAB vp fragment in V. parahaemolyticus M0605 likely located in a different site of a pVA1-type plasmid among them. The site was close to the insertion site of the solitary ISVal1 in pVA1. Note that since most of the reported genomes were unfinished, there is a possibility that there are undetected novel variants lying in the undetermined gaps.

Antibiotic Resistance
Antibiotic resistance of Vp 2S01 and Vc 3S01 were detected. The resistance gene profiles of the two strains were also Frontiers in Microbiology | www.frontiersin.org  revealed. Vp 2S01 harbored two antibiotic resistance genes tet(35) and tet(34) on chromosome I, while Vc 3S01 contains tet(35) on chromosome I and sul2, strAB, tet(A), strB-2 (extra copy) and floR on pVCGX2. The results of disk diffusion test showed that Vp 2S01 was only susceptible to florfenicol. It was intermediate to ceftriaxone, nitrofurantoin, norfloxacin. Significantly, Vc 3S01 was resistant to a wider spectrum of antibiotics as being resistant or intermediate to all test antibiotics (Table S4).

DISCUSSION
We compared a VP AHPND and a VC AHPND isolate from the same AHPND-affected pond. They displayed a similar pathogenicity and both contained the pirAB vp genes carried by plasmids that are highly homologous to pVA1. This study reveals the dissemination of the hazardous genes in V. campbellii, which is likely due to interspecies horizontal gene transfer. *Reference VC numbers were described by Seitz and Blokesch (2013).
Although VP AHPND is almost the only pathogen known to cause AHPND, studies have indicate that non-parahaemolyticus AHPND-causing Vibrio is emerging. Indeed, certain V. harveyilike, V. owensii, and V. campbellii strains have been detected to cause AHPND and to be pirAB vp -positive (Kondo et al., 2015;Liu et al., 2015;Dong et al., 2017a;Han et al., 2017;Xiao et al., 2017). However, only a limited number of the isolates were characterized and compared. The VC AHPND we previously isolated displays a similar level of pathogenicity to VP AHPND . Immersion challenge for L. vannamei shrimp demonstrates that Vc 3S01-infected shrimp present similar mortality, AHPND manifestations and pathology to Vp 2S01infected shrimp. It seems that the pathogenicity encoded by pirAB vp is independent from host bacteria within closely related Vibrio species. Remarkably, the pirAB vp genes may be not originated from V. parahaemolyticus. A recent study has demonstrated that non-virulent V. parahaemolyticus becomes VP AHPND via acquiring a plasmid named pVA1 that expresses a deadly toxin Pir vp (Lee et al., 2015). Additionally, the spread of the VP AHPND via diseased animals and contaminated water accelerates the pandemic of this disease. Similarly, other Vibrio species also could become pathogenic by acquisition of this plasmid. The prevalence of VP AHPND may suggest either they have better capacity of colonization or they are the first pathogenic host bacterium of this plasmid.
The pirAB vp -bearing plasmids pVPGX1 and pVCGX1 are highly homologous to pVA1, suggesting the occurrence of horizontal transfer of the pVA1-type plasmid. The pVA1-type plasmids with or without pirAB vp genes have been found in many Vibrio species (Xiao et al., 2017). Plasmid is an important factor driving the dynamic gene flow and promoting the fitness of host bacteria (Laurenceau et al., 2013;Seitz and Blokesch, 2013;Matthey and Blokesch, 2016). Plasmid can be acquired mainly through transformation or conjugation. It has been reported that the pVA1 plasmid contained a set of conjugative transfer genes, which suggests that pVA1 might be self-transmissible (Lee et al., 2015). Meanwhile, homologs of genes encoding a DNA-uptake machinery reasonable for natural competence of V. cholera (Seitz and Blokesch, 2013) were also found in the VP AHPND and VC AHPND isolates. Given that the two isolates were from the same origin and the prevalence of VP AHPND , there is a possible scenario that the pVA1-type plasmid pVCGX1 in VC AHPND was originally acquired from VP AHPND , via either self-mediated conjugation or uptake of the free plasmids released by dead VP AHPND cells. Interestingly, the Vc 3S01 contains 4 heterogeneous plasmids. It's unusual that so many different plasmids exist in one Vibrio cells. It may be due to the strain Vc 3S01 has a strong capability to obtain other plasmid. However, whether these transfer genes facilitate the intra-species dissemination of pirAB vp still awaits investigation. Moreover, Vc 3S01 is also "armed" with multiple antibiotic resistance genes, which will bring difficulties to control its future spread. Therefore, the transfer of these genes into a new host bacterium not only increases the complexity of causative agents, but also leads to a potential threat with no drug for treatment.
Despite the synteny of the carrier plasmids, the pirAB vp genes have dynamic contexts. We assumed the configuration showed in Figure 4C was resulted from the insertion of a new ISVal1 followed by recombination. We subsequently analyzed the flanking sequences of relevant ISVal1 elements to identify the recombination event using the method proposed by Partridge (2011) and found that the right part might have gone through an inversion event after the insertion of a new ISVal1. This right part was bounded by the oppositely oriented middle ISVal1 and right ISVal1. A 9-bp left flank (5 ′ -TACGGCATT-3 ′ ) of the middle ISVal1 was reverse-complement to that of the right ISVal1 (5 ′ -AATGCCGTA-3 ′ ). If we inverse the current form of this region, the 9-bp direct repeats flanking the middle ISVal1 could be restored and the direction of synteny to pVA1 would be consistent to that of the left part. Thus, an evolution process of pirAB vp contexts was deduced. A new ISVal1 inserted into the original pirAB vp contexts, generating 9-bp direct repeats (5 ′ -TACGGCATT-3 ′ ) and a sub-region that was also bounded by two opposite ISVal1. Then inversion of the sub-region occurred, resulting in the current form.
The pirAB vp context initially revealed on pVA1 is bounded by two identical but inversely oriented IS elements, ISVal1. Mobile genetic elements such as IS elements, transposons, integrons and genomic islands are important factors shaping genetic contexts of antimicrobial resistance genes and virulence genes (Partridge, 2011;Stokes and Gillings, 2011). Inversely oriented IS elements close to each other are potential targets for homologous recombination (Partridge, 2011). Unsurprisingly, an inversion form of pirAB vp context was found on pVPGX1. It has been suggested that the natural acquisition or deletion of the pirAB vp genes is due to transposition or homologous recombination (Lee et al., 2015). However, we prefer the former theory since the recombination results in inversion instead of excision or integration of pirAB vp contexts. The two ISVal1 might have formed a composite transposon. Moreover, it seems that ISVal1 is also a factor that complicates the pirAB vp context since the more complex configuration on pVCGX1 is very likely due to the insertion of a new ISVal1 followed by recombination. Therefore, we propose that ISVal1 plays a role not just in the translocation of the pirAB vp genes but also in the modulation of their contexts.
TCBS plate is currently used as a simple tool in many shrimp farms for monitoring and forewarning the V. parahaemolyticus-associated risk that may cause AHPND, as most V. parahaemolyticus strains develop green colonies (Non-sucrose-fermenting bacterial colonies are covered by the green color). This method works for V. campbellii as well. Indeed, neither Vp 2S01 nor Vc 3S01 contains genes that encode the sucrose hydrolase, such as scrB gene (Reid and Abratt, 2005). Other Vibrio species, like V. alginoluticus, V. cholerae carry the sucrose hydrolase genes and develop yellow colonies. A V. owensii strain from a shrimp farm suffering AHPND in Haiyang of Shandong Province in 2017 was also tested as pirAB VP positive. The strain was tested on TCBS plate and developed a yellow colony (unpublished results). Therefore, there is a potential risk of negative detection of pathogenic strains when using the TCBS plate method given the increasing intra-species dissemination of the pirAB VP genes. We here suggest both green and yellow colonies should be tested under such circumstance.
AHPND has caused severe production collapses and heavy economic losses in Asia and Latin America. (Zhang et al., 2012;FAO, 2013;De Schryver et al., 2014;Nunan et al., 2014;de la Pena et al., 2015;Lee et al., 2015). Actions to bring the disease to an end are in urgent need. This study demonstrates the dissemination of the pirAB vp genes in V. campbellii and thus sheds light on the molecular epidemiology of the virulence genes. Moreover, acquisition of the pVA1-type, pirAB vp -bearing plasmids in diverse Vibrio species increases the complexity of causative agents of AHPND and their potential threat to the shrimp industry. Therefore, our study provides timely information for better understanding of the causes, epidemiological features of AHPND, as well as developments of response measures and prevention and control strategies.

AUTHOR CONTRIBUTIONS
XD and DB designed and conducted the study, performed most of the experiments, and as well as HW wrote the manuscript. PZ, QY, XW, MC, CG, and WW performed the biological experiments. ZL assembled preliminary sequences and analysis. HW, QY, GX, XW, YZ, and QW discussed the results and modified the manuscript. JH designed the study and wrote the manuscript. All authors reviewed the manuscript.