Characterization of the Virulency of Vibrio spp. in Giant Tiger Shrimp ( Penaeus monodon ) from Peninsular Malaysia

Nowadays, bacterial diseases caused by Vibrio spp. pose a signi ﬁ cant obstacle to aquaculture growth, affecting socioeconomic development. The presence of pathogenic Vibrio spp. are gaining attention due to widespread mortality in ﬁ sh and shell ﬁ sh worldwide. This study investigates the distribution of virulence genes and their pathogenicity by experimentally infecting a healthy group of cultured shrimp, speci ﬁ cally Penaeus monodon . A total of 225 Vibrio spp. isolated from 210 cultured shrimp from various geographical regions in Peninsular Malaysia were examined, resulting in 13 Vibrio spp. being identi ﬁ ed and characterized. According to virulence gene assessment, only eight of the 11 virulence genes were detected in the isolates, including chiA (89 % ), ﬂ aC (74 % ), tlh (72 % ), toxR (70 % ), luxR (40 % ), pirA (11 % ), pirB (11 % ), and hlyA (0.4 % ). Other virulence genes, such as tdh , trh , and ctxA , were not found in any of the isolates (0 % ). Then, three Vibrio isolates, including V. parahaemolyticus S12-3, V. campbellii S10-4, and V. rotiferianus S24-4, were selected for pathogenicity test based on highest number of virulence genes in each species. These species were subjected to immersion challenge test against postlarvae (PL) P. monodon approximately PL-20 with three different concentrations of 1 × 10 6 , 1 × 10 7 , and 1 × 10 8 CFU/mL. The V. parahaemolyticus S12-3 was determined to have lethal concentration 50 (LC 50 ) 1 × 10 5 CFU/mL after 168 hr of exposure. The immersion challenge test resulted in a pale hepatopancreas and empty guts in P. monodon . Histopathological examination showed sloughing of epithelial cells in the hepatopancreatic tubule, hemocytic in ﬁ ltration, massive vacuolation, and loss of hepatopancreatic tubule structure. Overall, the study demonstrated the virulence and pathogenicity of V. parahaemolyticus S12-3 toward P. monodon , which could be associated with its virulence properties. Furthermore, the current ﬁ ndings will be useful in developing microbiological risk assessments for shrimp farming and aquaculture management.


Introduction
Shrimp aquaculture contributed to 15% of all worldwide traded seafood products [1] According to FAO [2], the shrimp industry contributes USD 250 billion to regional, national, and local economies.The main producers of giant tiger shrimp (Penaeus monodon) and whiteleg shrimp (Penaeus vannamei) are China, Vietnam, India, Thailand, and Indonesia, which contributing production values of USD 5.59 billion and USD 26.7 billion [3,4].However, the Asia-Pacific region's shrimp industry, as the world's leading producer of aquaculture products, has been affected by emerging diseases, resulting in high mortality rates and economic losses.The common diseases such as luminous vibriosis, white spot disease (WSD), yellow head disease (YHD), Taura syndrome (TS), and acute hepatopancreatic necrosis disease (AHPND) have devastated the region's shrimp aquaculture, resulting in the shrimp industry's collapse [5].Therefore, maintaining the animals' health is crucial to prevent the outbreak.
Vibriosis is a common bacterial disease that affects a wide range of marine fish and shellfish.It has been reported that vibriosis caused severe production losses in shrimp farms, with mortality rates of up to 100%, particularly in postlarvae and juvenile populations across all producing countries [6].Vibrio spp.such as V. alginolyticus, V. anguillarum, V. harveyi, V. vulnificus, and V. parahaemolyticus are the most common species that cause vibriosis in shrimp [7].Furthermore, V. parahaemolyticus is one of the several causative agents of shrimp disease that has become more prevalent as global shrimp demand and production have increased [8].The presence of pathogenic bacteria in the global marine environment lead to high mortality rates and can result in significant economic losses.The bacteria enter into aquaculture system through contaminated water, feed, and equipment [9].Therefore, a good management practices should be implemented to promote a healthy and productive industry, particularly shrimp industry.
There are a variety of virulence genes that encode virulence factors that contribute to Vibrio strains' pathogenicity [10].The virulence factors enable pathogens to infect and harm hosts by allowing adhesion and entry, establishment and multiplication, evading host defenses, causing host damage, and eventually leaving the host [11].Besides, five major virulence factors, which play a role in microbial pathogenesis, found in Vibrio such as capsular polysaccharides, adhesive factors, cytotoxins, lipopolysaccharides, and flagella [9,12].Furthermore, these virulence factors may be required for virulence against various hosts because vibrios infect a diverse range of aquatic species, including fish, shrimp, and mollusks [13].According to Menezes et al. [14], the aquaculture environment provides a suitable condition for virulent lineages of Vibrio.This is primarily due to the high level of nutrients and the presence of antibiotics, providing the optimum environment for growth and development of the microorganisms.
There has been little research on virulence and pathogenicity of several Vibrio spp. that cause vibriosis in shrimp, particularly in Malaysia.This makes it difficult to identify, treat, and prevent, causing significant losses in the shrimp farming industry.Therefore, a study on virulence genes profiling and their pathogenicity was conducted to provide a better knowledge of their potential virulence.In addition, further study is required to understand the differences in pathogenicity across Vibrio spp.and to develop effective management techniques for vibriosis in shrimp.

Collection of Vibrio spp.
A total of 225 Vibrio isolates that were previously obtained from shrimp were used.The isolates were identified using pyrH gene in previous experiment [15] as V. parahaemolyticus , and Photobacterium damselae (n = 7).The isolates were streaked onto tryptic soy agar (TSA) (Oxoid, Hampshire, UK) supplemented with 1.5% NaCl and incubated at 30°C for 24 hr.Then, the single colony on TSA was further subcultured on TCBS agar.

Virulence Genes Assessment.
A total of 11 virulenceassociated genes, including pirA, pirB, chiA, luxR, flaC, hlyA, toxR, ctxA, tlh, tdh, and trh, were amplified by polymerase chain reaction (PCR), and the sequence of primers used were shown in Table 1.The PCR mixtures were performed in a final volume of 25 μL containing 2.5 μL of the DNA template (10-100 ng) that was mixed with 12.5 μL of PCR MasterMix (First Base, Kuala Lumpur, Malaysia) comprised of 60 U/mL of Taq DNA polymerase, 3 mM MgCl 2 , 400 µM dNTP mix, and 10 µM each forward and reverse primer.
The amplification was performed under the following conditions: initial denaturation at 95°C for 5 min, followed by 30 cycles of 95°C for 1 min; 48.9°C for 1 min (chiA), 50°C for 1 min (luxR), 60°C for 1 min (hlyA), 55°C for 1 min ( flaC) and 72°C for 1 min, and a final extension of 72°C for 10 min.The amplification of tlh, trh, and tdh was performed under the following conditions: initial denaturation at 94°C for 3 min, followed by 30 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min, and a final extension of 72°C for 10 min.The amplification of pirA and pirB was performed under the following conditions: initial denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 58.5°C for 30 s, and 72°C for 30 s, and a final extension of 72°C for 10 min.For ctxA and toxR, the conditions were initial denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 45 s, 60°C for 40 s (ctxA), 52°C for 45 s (toxR), and 72°C for 1 min, and a final extension of 72°C for 10 min.Then, the PCR amplifications were carried out using a PCR thermocycler (Bio-Rad, California, USA).The PCR products were visualized with a gel documented system (Acinterlab, Miami, USA) after gel electrophoresis (70 V, 60 min) using 1.5% agarose gel electrophoresis that had been preadded with FloroSafe DNA Stain (First Base Laboratories).

Acclimatization of Giant Tiger Shrimp (P. monodon).
A 20-day postlarvae (PL-20) cultures with 0.09 AE 0.03 g in weight were obtained from a local commercial shrimp hatchery.The shrimp were acclimatized in a 500-L tank with constant aeration for 7 days.Approximately 30% of the seawater was replaced, and water quality measures were evaluated on a daily basis.The mean AE SD of water parameters, including pH, temperature, salinity, and dissolved oxygen, were determined a using a handheld YSI meter (YSI Incorporation, Ohio, USA).During the entire experiment, the shrimp were fed with commercial feed containing 35% crude protein three times daily [23].Briefly, five colonies formed on TCBS agar were randomly picked and inoculated into 1 L of TSB supplemented with 1.5% NaCl and incubated at 30°C for 24 hr, with a constant shaking at 150 rpm.Then, serial dilutions were performed up to 10-fold and 0.1 mL of TSB from each dilution was plated on TSA in triplicate, and further incubated at 30°C for 24 hr.The colony-forming units per milliliter (CFU/mL) was calculated using standard plate count.

Pathogenicity Study.
For the pathogenicity test, a total of 540 PL of P. monodon were randomly distributed into 10 L of tank.The shrimp were divided into three groups (three groups × three bacterial dilutions × 15 shrimp/tank) of bacterial isolates and three group of control (15 shrimp × three replicates).Each of the treatment was conducted in triplicate (n = 3).The experiment was conducted using immersion challenge test as described by Tran et al. [24] with some modifications.The bacterial cells were harvested from the overnight incubation by centrifugation at 7,000 rpm for 10 min.Then, the shrimp were immersed into fresh bacterial suspension for 15 min.Next, the shrimp were transferred into the experimental tank with bacterial suspension to obtain final concentration of 1 × 10 6 , 1× 10 7 , and 1 × 10 8 CFU/mL.Control shrimp were not exposed to any of the isolates.The shrimp were maintained with commercial shrimp feed at 5% body weight throughout the period.
Moribund and dead shrimp were removed daily from the tanks.During the experiment, all tanks were aerated with zero water exchange for the duration of 168 hr.In all challenges, the moribund shrimp were anesthetized using MS-222 (Western Chemical Inc., Ferndale, WA, USA) at a concentration of 100 ng/mL [25].The abnormalities and mortalities of shrimp were observed and expressed as the cumulative mortality.The lethal concentration 50 (LC 50 ) was calculated using probit analysis with SPSS version 25.The freshly dead shrimp were subjected to bacterial isolation and identification [26].
2.6.Histopathology Analysis.Histological analysis was performed to study the pathologic changes.The hepatopancreas of shrimp was taken for the analysis at the end of the experiment.Shrimp were immediately fixed in 10% (v/v) phosphate-buffered formalin composed of 100 mL of 40% formalin, 900 mL of distilled water, 4 g/L of NaH 2 PO 4 , and 6.5 g/L of Na 2 HPO 4 for 24 hr.Then, the shrimp were preserved in 70% ethanol until further processing [27,28].The preserved samples were submitted to the Veterinary Histopathology Laboratory at Universiti Putra Malaysia for further processing.Briefly, the samples were trimmed and were routinely processed and embedded using the paraffinembedded technique.These tissues were sectioned at 4 µm thickness, dried overnight, and were routinely stained using hematoxylin and eosin (H&E) staining.All tissue sections were examined under a light microscope to describe and compare the histopathological features.

Statistical Analysis.
The statistical significance of data was measured using the analysis of variance (ANOVA) test.A p-value <0.05 was set as a statistically significance.Mean, standard deviation, and graph for cumulative mortalities were constructed using Microsoft Excel.
According to the findings, 99% of the isolates included possessed at least one virulence gene, whereas 1% do not possessed any virulence genes.The highest number of Vibrio isolates harbored seven out of 11 virulence genes, including pirA, pirB, tlh, flaC, toxR, chiA, and luxR.In addition, the majority of the Vibrio isolates had four virulence genes (44%), followed by 20% with three virulence genes, 16% with five virulence genes, 7% with six virulence genes, 7% with two virulence genes, and 4% with one virulence gene.Notably, no Vibrio isolates possessed virulence genes such as tdh, trh, or ctxA (Table S1).

Pathogenicity Study.
In pathogenicity study, three isolates were selected based on the number of highest virulent genes present in each isolate, as shown in Table S1.The isolates were V. parahaemolyticus S12-3, V. campbellii S10- Aquaculture Research 4, and V. rotiferianus S24-4.Since these isolates carried the highest number of virulence genes, therefore, these isolates were selected for the pathogenicity test.Table 2 compares the percentage of mortality from pathogenicity tests of V. parahaemolyticus S12-3, V. campbellii S10-4, and V. rotiferianus S24-4.The findings demonstrate that V. parahaemolyticus S12-3 isolate was the most virulent with LC 50 1 × 10 5 CFU/mL.On the other hand, the LC 50 for V. campbellii S10-4 was 1 × 10 13 CFU/mL and V. rotiferianus S24-4 was 1 × 10 11 CFU/mL.As shown in Table 2, at higher concentration of V. parahaemolyticus S12-3 (1 × 10 8 CFU/mL), the first mortality began as early as 12 hr (11%) and reached 50% of the mortality at 48 hr.The shrimp reached 100% mortality within 72 hr.In comparison to 1 × 10 8 CFU/mL, it takes a few hours for shrimp challenged with V. parahaemolyticus S12-3 to die at a concentration of 1 × 10 7 and 1 × 10 6 CFU/mL.The shrimp obtained a complete mortality (100%) at 108 hr when challenged with 1 × 10 7 CFU/mL and 132 hr when challenged with 1 × 10 6 CFU/mL.There was a significant difference (p<0:05) from 0 to 108 hr when the shrimp were challenged with 1 × 10 7 CFU/mL of V. parahaemolyticus S12-3.
Following daily monitoring, the shrimp between control and challenged groups were observed for the clinical signs.The clinical signs observed in moribund shrimp were similar to those observed in infected shrimp from farms.The common clinical signs include lethargy and abnormal swimming behavior.In addition, infected shrimp may show a decrease in feeding activity and a lack of interest in food, which contribute to decreased growth and weight gain (Figure 4(a)).This would give a negative impact on their overall health and performance.Aside from that, hemorrhaging was characterized by red or brown discoloration or appendages caused by bleeding beneath the exoskeleton, which can indicate a serious bacterial infection (Figure 4(b)).The affected area was red and swollen due to the inflammation.In addition, the exoskeleton also became soften and brittle, making the shrimp more vulnerable to damage and disease.The gross lesion displayed by the challenged shrimp includes an empty stomach, as well as white and atrophied hepatopancreas (Figure 4(c)) compared with the normal shrimp (unchallenged) with large pigmented hepatopancreas (Figure 4(d)).At the end of the experiment, reisolation of the bacteria indicated that the growth of the colony was the same as the colony isolated from the farms (Figure 4(e)-4(g)), and the Vibrio spp.were confirmed by PCR.

Histopathology Analysis.
The histopathological features of hepatopancreas were analyzed at the end of the experiment.Histopathological examination of the normal shrimp hepatopancreas revealed no histopathological changes.In general, the hepatopancreatic tubules were intact, with relatively uniform size and a large central lumina.The tubules were lined by viable and healthy B and R cells, whereby the B cells showed a single large vacuole, while the R cells showed numerous small intracytoplasmic vacuoles (Figure 5(a)).In contrast, few abnormalities were noted in the hepatopancreas of PL infected with pathogenic Vibrio spp.The hepatopancreatic tubules were of various sizes and usually distant from one another.The lumens were usually small, largely incriminated to hepatopancreatic epithelial changes.Most of the B and R cells lacked intracytoplasmic vacuoles.Instead, their cytoplasm showed intracytoplasmic eosinophilia, while the nuclei were round, basophilic, and centrally located.Stacking of these epithelial cells was observed, accompanied by elongation, leading to prominence that partially occluded the hepatopancreatic tubular lumen (Figure 5(b)).
Other changes included the sloughing of epithelial cells (Figure 5(c)) and enlarged of nuclei (Figure 5(d)), leading to observation of individual cells or cellular debris in the lumen of the hepatopancreas.Severe and a more advanced histopathological changes included structural loss, largely due to severe necrosis of the epithelial cells, as well as rupture of the hepatopancreatic tubules.Regardless of the lesion severity involving the hepatopancreatic tubules, hemolytic infiltrates were seen in moderate to high number (Figure 5(e)).Moreover, some hepatopancreatic tubules showed presence of epithelial cells that exhibited vacuolation resulting from inflammatory reaction (Figure 5(f )).

Discussion
Vibriosis is a major disease problem in shrimp farming resulting in high mortality and significant economic losses in all producing countries [29].Vibrio spp.are one of the most important bacterial pathogens of cultured shrimp, causing a variety number of diseases and up to 100% 6 Aquaculture Research  Note: Mean that does not share the same letter shows a significant difference (p<0:05).
Aquaculture Research mortality rates [30].The most pathogenic Vibrio spp.found in shrimp, including V. harveyi, V. penaeicida, V. parahaemolyticus, and V. vulnificus, which possessed various important virulence factors [6,31].Therefore, identification of virulence factors is essential for evaluating bacterial pathogenicity because these factors allow bacteria to infect and damage hosts [32].In this study, we demonstrated the presence of virulence genes associated with pathogenic strains that may contribute to the pathogenicity of Vibrio spp., which were previously isolated from cultured shrimp in Peninsular Malaysia.
The main factor in Vibrio pathogenicity is virulence factors, which are expressed by virulence genes [10].Vibrio spp.infect and damage the host through these five elements of virulence factors such as capsular polysaccharide, adhesion factor, cytotoxin, lipopolysaccharide, and flagellum [9].Then, they allow pathogens to adhere, enter, establish, and multiply while evading the host's immune defense system [11].Furthermore, Vibrio can acquire atypical virulence genes, which can further increase its pathogenicity [33].It has been discovered that these atypical virulence genes can be acquired from the environment or from other bacteria through horizontal gene transfer (HGT) [34].In addition, other factors such as global climate change, antibiotics, heavy metals, and environmental eutrophication might affect bacteria stress response mechanism, which could induce mutagenesis and HGT, and ultimately increasing their pathogenicity and antibiotic resistance [35][36][37].The observation through the gross lesion also serves as a sign of the pathogen's virulence.More virulent pathogens, for example, may create larger and more severe lesions, whereas less virulent pathogens may cause smaller and less severe lesions [38].As a result, it is essential for comprehending disease mechanisms and developing disease control.In this study, chiA was found to be the most prevalent gene among the isolates (89%).In addition, chiA was widely distributed in V. parahaemolyticus compared to another Vibrio spp.The result was in concordance with the findings reported by Mohamad et al. [39] where all 63 isolates of Harveyi clade, including V. parahaemolyticus, V. alginolyticus, V. harveyi, and V. campbellii, possessed virulence gene of chiA (100%).Another study by Ruwandeepika et al. [17] also revealed that 48 isolates of Harveyi clades, including 47 isolates of V. harveyi and one isolate of V. campbellii from diverse range of hosts and geographical locations, were found to carry chiA gene.The chiA gene is associated with the production of chitinases, which are the enzymes capable of degrading chitin.Chitin is a substrate that can be found abundantly in water, and the presence of chiA gene in Harveyi clades suggests that these bacteria may have the ability to degrade chitin.The ability to utilize chitin could promote pathogen's survivability in nutrient-scarce environment, while contributing to their pathogenicity by altering host's immune response [40,41].
Flagella play an important role in infecting host because it increases the motility of bacteria for colonization or act as adhesive [42].Other than chiA, this study revealed that flaC also possessed a high number of percentage (74%) where it was dominantly found in V. parahaemolyticus.On the contrary, a study conducted by Bai et al. [18] revealed that most of the flaC was detected in V. harveyi.However, the flaC was also detected in various Vibrio spp.such as V. parahaemolyticus, V. alginolyticus, V. anguillarum, V. campbellii, V. fischeri, V. fluvialis, V. mimicus, and V. natriegens, aligning with the Vibrio spp.identified in our study.In addition, another study discovered that 68% of the Vibrio isolates possessed for flaC [39].Other virulence gene such as thermolabile hemolysin gene, tlh, has previously been used to identify V. parahaemolyticus [22].However, it has been reported that flaC not only found in V. parahaemolyticus but also could be found in V. alginolyticus, V. harveyi, V. fischeri, V. mimicus, V. natriegens, V. proteolyticus, V. anguillarum, and V. vulnificus [43].In this study, 72% of the isolates possessed for tlh, including V. parahaemolyticus, V. alginolyticus, V. campbellii, V. rotiferianus, V. communis, V. owensii, V. brasiliensis, and Vibrio spp.The findings were supported by Amatul-Samahah et al. [44] demonstrating that all of Vibrio strains were positive for tlh (100%).Furthermore, Mohamad et al. [39] revealed that more than 50% of the Vibrio isolates possessed tlh.In addition, a study by Joshi et al. [45] also reported to be positive for tlh on V. parahaemolyticus.
The toxR is a transmembrane transcription regulator that regulates the expression of virulence gene in V. cholerae [12].However, recent studies found that toxR is present in other vibrios, including V. parahaemolyticus, V. fischeri, V. vulnificus, V. alginolyticus, V. hollisae, V. mimicus, V. fluvialis, V. harveyi, V. anguillarum, and Photobacterium spp., which aligned with this study where 70% of the Vibrio isolates possessed toxR gene [17].In addition, according to Asgarpoor et al. [46], 12 out of 70 shrimp (17.1%) were possessed for toxR gene.Furthermore, the occurrence of positive toxR in V. parahaemolyticus gene was approximately in line with findings identified by Abd-Elghany and Sallam [47] who discovered that 14% of shrimp samples were positive for toxR.Moreover, a study by Hassan et al. [48] found that 19% (38/200) of retailed shellfish samples were possessed for toxR gene.
According to the study's findings, 11% of the isolates possessed pirA and pirB, respectively.The AHPND-causing strain is an opportunistic marine shrimp pathogen that harbors pirA and pirB genes [49].Originally, pirA and pirB are found in Photorhabdus luminescens bacteria and responsible for encoding pirAB toxin, which indicates the insecticidal activity against Spodoptera exigua larvae [50].The pirA and pirB genes are located on the self-transmissible plasmid of AHPND causing V. parahaemolyticus strains [51].A study conducted by Chonsin et al. [52] showed that 10.84% (9/83) of V. parahaemolyticus isolates were positive for pirA and pirB.Similarly, there is also a positive finding for pirA and pirB genes of V. parahaemolyticus isolates in 9.9% shrimp and 0.7% molluskan shellfish have been reported from Mekong Delta in Vietnam [53].Contrary to the expectation, our findings showed that V. campbellii was dominantly possessed pirA and pirB than V. parahaemolyticus from the prior study.There are several factors that may contribute to the contradictory results, including species variations, regional variation, and the level of the virulence [54].However, a study by Dong et al. [55] demonstrated the presence of pirA and pirB found in V. campbellii, which is consistent with our findings.
The virulence of several pathogenic bacteria has also been associated with quorum sensing, a form of bacterial cell-to-cell communication involving small signaling molecules [56].Therefore, luxR is important as key regulator of virulence in pathogenic bacteria.It regulates gene expression in response to other bacteria's signals, allowing for coordinated virulence and immune evasion.Thus, luxR plays an important role in bacterial pathogenicity by regulating the expression of genes involved in infection and immune evasion [54].A study by Ruwandeepika et al. [17] discovered that 48 isolates from the Harveyi clade carried luxR gene.In addition, all 63 isolates of Harveyi clades from Mohamad et al. [39] possessed typical virulence genes of luxR (100%).Both studies showed a high percentage of luxR.In contrast, our finding revealed a low percentage of the isolates that possessed virulence gene of luxR (40%) compared to the previous study.Our findings also were in close agreement reported by Deng et al. [33] where only 12.6% of the Vibrio strain possessed for hlyA gene, which among the lowest of virulence gene found in their study.
However, none of the isolates were detected for the presence of tdh, trh, and ctxA genes.The tdh and trh genes are vital for the virulency of pathogen, as these genes encode the pore-forming proteins involved in the invasiveness of pathogen [44].These genes were not detected in this study, most likely because these isolates were isolated from an environmental source as reported by Xie et al. [57].This implies that there is no association between these genes and pathogenic Vibrio in humans, indicating a low potential risk for human health.Our findings appear to be well supported by previous studies.A finding by Chonsin et al. [52] showed that none of the tested samples contained tdh and trh genes.Similarly, Joshi et al. [45] reported that none of their Vibrio isolates carrying both tdh and trh genes.A study by Guardiola-Avila et al. [7] did not discover the ctxA gene, which is consistent with our findings.In addition, several other researchers have also discovered that this gene may not always present in the 10 Aquaculture Research environment [58].Therefore, it is essential to emphasize that the strains isolated in our study, even without the ctxA gene, could pose a significant health risk to the population, as they are capable of causing potentially life-threatening diarrheal diseases [59].
The virulence factors may be essential for infecting toward different hosts, as Vibrio spp.have the capacity to infect a broad spectrum of aquatic organisms [32,60].While the relationship between the number of virulence genes and pathogenicity in Vibrio is complicated, the acquisition of atypical virulence genes may enhance the ability of the bacteria to infect specific hosts [33].The virulency of the isolates was further assessed by conducting challenge tests with PL of P. monodon to determine the pathogenicity of the Vibrio isolates.Despite the presence of typical virulence genes in all isolates, there was considerable variation in their virulence levels.In addition, some of the isolates causing high mortality did not have any atypical virulence genes, while others causing low mortality have atypical virulence genes [17].
In the pathogenicity test, three Vibrio spp., including V. parahaemolyticus S12-3, V. campbellii S10-4, and V. rotiferianus S24-4, were selected due to the highest number of virulence genes possessed in each of the isolates.The findings suggest that virulence genes are transferred among Vibrio spp.and that acquiring virulence genes would increase the virulency of the Vibrio to hosts [33].In aquatic environments, Vibrio spp.are a significant reservoir of potential virulence and bacterial DNA material exchange through HGT [61].
According to the findings, when the shrimp were challenged with V. parahaemolyticus S12-3, it resulted in 100% of mortality within 72 hr at 1 × 10 8 CFU/mL.The results obtained were similar with a study by Thakur et al. [62] in which the shrimp were challenged with V. parahaemolyticus that showed 100% of mortality within 72 hr.When the shrimp were challenged with V. rotiferianus S24-4 and V. campbellii S10-4 with the same concentration of 1 × 10 8 CFU/mL, the percent mortality observed was 11% and 2%, respectively.In comparison to Thakur et al. [62], V. parahaemolyticus isolated from P. monodon in India's West Coast had a greater LC 50 of 5.99 × 10 5 CFU/mL as opposed to 1 × 10 5 CFU/mL in our findings.On the other hand, V. campbellii S10-4 in the present study had a higher LC 50 of 1 × 10 13 CFU/mL in contrast to V. campbellii strain as reported by Nurhafizah et al. [63] which had LC 50 of 6.0 × 10 8 CFU/mL.The V. parahaemolyticus S12-3 has a high mortality rate and can cause death in a short time.In addition, V. parahaemolyticus S12-3 has the lowest LC 50 compared to V. rotiferianus S24-4 and V. campbellii S10-4.Therefore, this suggest that the presence of virulence genes plays an important role in the pathogenicity of Vibrio spp.[33].
The LC 50 values may differ due to the virulence properties of the two Vibrio spp., as well as the state and condition of the experimental shrimp employed.Wang et al. [64] stated that factors such as bacterial concentration, exposure period, and infection method were identified as contributing to mortality.As for example, a study conducted by Joshi et al. [45] revealed that by performed immersion challenge on shrimp with V. parahaemolyticus at the concentration of 10 8 CFU/mL led to 100% of mortality within 24 hr.However, our study, employing the same method, concentration, and species, demonstrated 100% of mortality at 72 hr, indicating a longer duration for mortality compared to findings by Joshi et al. [45].Another study by Le Hong et al. [65] revealed that when the shrimp challenged with V. campbellii at the concentration of 10 8 CFU/mL, they exhibited mortality less than 20% at 48 hr.In contrast, our findings observed a lower mortality rate (2%) at 72 hr.
The findings revealed that although each of the isolates possessed the same number of the virulence factors (7/11), there were variations in their virulence level.The differences of pathogenicity were depended on species tested and on the strain characteristics.Moreover, the study observed that mortality resulting from challenge test depended on the doses and species [64].In addition, global climate change, antibiotics, heavy metals, and nutrient pollutants have been reported to induce the expression of virulence genes and increase the pathogenicity [66].According to Guijarro et al. [67], V. coralliilyticus exhibits 16-fold elevation in virulence factors related to motility, host degradation, secretion, resistance to antimicrobials, and transcriptional regulation when the temperature increased from 24 to 27°C.
During challenge test, the affected shrimp can be identified by observing both physical and behavior of shrimp.On a regular basis, there was a decrease in shrimp appetite, empty gut, and a lot of leftover feed on the basis of maintenance.Furthermore, the larvae looked weak and sluggish with the head constantly facing up and uncontrolled movements.Moreover, the shrimp tail was necrotizing, melanosis on the skin, the body of the shrimp looked pale while swimming legs, and telson and uropod were become reddish [68].The symptoms were comparable in our study where the symptom can be observed as early as 12 hr after being challenged with V. parahaemolyticus S12-3.Similar clinical signs have been recorded in shrimp with vibriosis when challenged with V. parahaemolyticus at the concentration of 1 × 10 6 CFU/mL [69].
Histopathology of the hepatopancreas revealed that the hepatopancreas was in various stages of degeneration after immersion challenge test.The findings were similar to the findings by Tran et al. [24] and Rowley and Pope [70].Based on histopathology analysis, the hepatopancreatic tubular epithelial cells have been changed and extended toward the lumen, resulting in cellular desquamation.Furthermore, there was a sloughing of hepatopancreatic tubular epithelial cells, hemocytic infiltration, and disorganized structure of hepatopancreas tubule that were characterized by massive vacuolation due to inflammatory response [71,72].Conversely, normal hepatopancreas showed a complete structure of hepatopancreatic tubular cells similarly with a study conducted by Nurhafizah et al. [63].

Conclusion
In conclusion, there were presence of multiple virulence genes in V. parahaemolyticus, V. rotiferianus, and V. campbellii.From the pathogenicity test, it was found Aquaculture Research that V. parahaemolyticus S12-3 was the most virulent with LC 50 1 × 10 5 CFU/mL.The study was supported by histopathological findings, which were typical after Vibrio spp.infection.The results from this study would provide valuable insights on Vibrio pathogenesis and will contribute in the development of vaccines, probiotics, immunostimulants, and ecological control systems in the aquaculture industry.Therefore, this study warrants a continuous long-term monitoring of the pathogens of shrimp, such as Vibrio spp., since its outbreak may have devastating impact on the Malaysian shrimp export industry in particular.

FIGURE 2 :
FIGURE 2: The distribution pattern of virulence genes detected in the isolates of Vibrio spp.

FIGURE 4 :
FIGURE 4: Clinical signs in P. monodon resulted from immersion challenge test of three Vibrio spp: (a) comparison between normal and challenged shrimp, (b) normal shrimp showed black uropod (blue circle), while challenged shrimp showed reddening in uropod (red circle), (c) infected hepatopancreas after challenged, (d) normal hepatopancreas after challenged, (e) the growth of V. campbellii on TCS agar after reisolation, (f ) the growth of V. rotiferianus on TCS agar after reisolation, and (g) the growth of V. parahaemolyticus on TCS agar after reisolation.All the isolates were confirmed by PCR.

TABLE 1 :
Virulence factors, sequence of primers, references, and expected amplicon size of target gene used in this study. hepatarius FIGURE 3: The distribution of virulence genes of Vibrio spp.Different colors represent different virulence genes study.

TABLE 2 :
Percentage of mortality observed from the pathogenicity test after 168 hr.