Vibrio Parahaemolyticus Strains Research Articles

Isolation and Characterization of Infection of Four New Bacteriophages Infecting a Vibrio parahaemolyticus Strain

Isolation and Characterization of Infection of Four New Bacteriophages Infecting a Vibrio parahaemolyticus Strain

Passive immunoprophylaxis with Ccombodies against Vibrio parahaemolyticus in Pacific white shrimp (Penaeus vannamei)

The Vibrio parahaemolyticus strain causing acute hepatopancreatic necrosis disease (AHPND) in shrimp secretes toxins A and B (PirAVp/PirBVp). These toxins have been implicated in pathogenesis and are targets for developing anti-AHPND therapeutics or prophylactics that include passive immunization. We have previously reported that Ccombodies (recombinant hagfish variable lymphocyte receptor B antibodies; VLRB) targeting PirBVp conferred protection against V. parahaemolyticus in shrimp when administered as a feed supplement. In this study, we screened a phage-displayed library of engineered VLRBs for PirAVp-targeting Ccombodies that were mass-produced in a bacterial expression system. We then introduced these Ccombodies into the diet of Pacific white shrimp (Penaeus vannamei) over a seven-day period. Subsequently, the shrimp were exposed to a challenge with V. parahaemolyticus. Mortality rates were then observed and recorded over the following seven days. Administering shrimp feed supplemented with Ccombodies at a high dose (100 mg per 100 g feed) reduced mortality in recipient animals (2.96-5.19%) statistically similar to mock-challenged control (1.48%), but significantly different from the Ccombody-deficient control (74.81%). This suggests that the Ccombodies provided strong protection against the bacterium. Feeding shrimp with a median dose (10 mg/100 g feed) gave statistically comparable low mortality (5.93-6.67%) as the high dose. Reducing the Ccombody dose to 1 mg/100 g feed showed variable effects. Ccombody A2 showed mortality (11.85%) significantly lower than that of the Ccombody-deficient group (74.81%), suggesting that it can effectively protect against the bacterial challenge at a low dose. Our results demonstrate the ability of the phage-displayed VLRB library to generate antigen-specific Ccombodies rapidly and simply, with the expression of high protein levels in bacteria. The protective effect provided by these Ccombodies aligns with our earlier results, strongly supporting the use of VLRB antibodies as a substitute for IgY in passive immunoprophylaxis against AHPND in shrimp.

Genomic Characterization of Bacillus pumilus Sonora, a Strain with Inhibitory Activity against Vibrio parahaemolyticus-AHPND and Probiotic Candidate for Shrimp Aquaculture.

Acute hepatopancreatic necrosis disease, caused by Vibrio parahaemolyticus strains carrying the pirA and pirB toxin genes (VpAHPND), has been causing great economic losses in Asia and America in the shrimp farming industry. Numerous strains are resistant to antibiotics. However, supplementation with probiotic antagonists has become a more desirable treatment alternative. Fourteen strains of microorganisms were assessed for their potential to inhibit VpAHPND in vitro activity. The bacteria with the highest activity were challenged with VpAHPND-infected Pacific white shrimp Litopenaeus vannamei. Furthermore, the genomic characteristics of probiotic bacteria were explored by whole-genome sequencing. We identified the Sonora strain as Bacillus pumilus, which possesses positive proteolytic and cellulolytic activities that may improve shrimp nutrient uptake and digestion. Challenge trials showed a low cumulative mortality (11.1%). B. pumilus Son has a genome of 3,512,470 bp and 3734 coding sequences contained in 327 subsystems. Some of these genes are related to the biosynthesis of antimicrobial peptides (surfactins, fengycin, schizokinen, bacilibactin, and bacilysin), nitrogen and phosphorus metabolism, and stress response. Our in vitro and in vivo findings suggest that B. pumilus Sonora has potential as a functional probiotic.

Effect of plasma-activated water on planktonic and biofilm cells of Vibrio parahaemolyticus strains isolated from cutting board surfaces in retail seafood markets.

This research aimed to analyze cutting board surfaces in seafood markets to find Vibrio parahaemolyticus, assess the isolates' ability to form biofilms, generate and evaluate characteristics of plasma-activated water (PAW), and compare the effect of PAW on planktonic and biofilm cells of the isolated V. parahaemolyticus strains. A total of 11V. parahaemolyticus strains were isolated from 8.87% of the examined cutting boards. Biofilm-forming ability was evaluated for these isolates at temperatures of 10°C, 20°C, and 30°C using crystal violet staining. Four strains with the highest biofilm potential were selected for further analysis. The pH of the PAW used in the study was 3.41±0.04, and the initial concentrations of hydrogen peroxide, nitrate, and nitrite were 108±9.6, 742±61, and 36.3±2.9µM, respectively. However, these concentrations decreased significantly within 3-4 days during storage at room temperature. PAW exhibited significant antimicrobial effects on V. parahaemolyticus planktonic cells, reducing viable bacteria up to 4.54 log CFU/ml within 20 min. PAW also reduced the number of biofilm cells on stainless steel (up to 3.55 log CFU/cm2) and high-density polyethylene (up to 3.06 log CFU/cm2) surfaces, although to a lesser extent than planktonic cells. PAW exhibited significant antibacterial activity against V. parahaemolyticus cells, although its antibacterial properties diminished over time. Furthermore, the antibacterial activity of PAW against biofilm cells of V. parahaemolyticus was less pronounced compared to the planktonic cells. Therefore, the actual effectiveness of PAW in seafood processing environments can be affected by biofilms that may form on various surfaces such as cutting boards if they are not cleaned properly.

Rapid and simultaneous detection of virulence factors of Vibrio parahaemolyticus by multiplex microfluidic-LAMP (MMLAMP)

Vibrio parahaemolyticus (VP) is an essential foodborne bacterium who can cause gastroenteritis. However, current detection methods generally still have the difficulty for quickly screening VP strains and accurately detecting VP. In this work, a sensitive and specific multiplex microfluidic-LAMP (MMLAMP) method was constructed to rapidly detect VP using a centrifugal microfluidic chip combined with LAMP. The system enables simultaneous detection of two hemolysin genes (tlh and trh) and one VP regulatory factor (toxR) in a single chip improving the accuracy for detecting VP. The MMLAMP system detected 2.42 × 104 copies/μL of the plasmid (tlh-pUC57), 4.44 × 103 copies/μL of the plasmid (trh-pUC57) and 4.86 × 104 copies/μL of the plasmid (toxR-pUC57) in less than 35 min, respectively. Meanwhile, MMLAMP system was successfully applied for VP detection in artificially contaminated samples, the LOD is 220 CFU/g, which is 10 times lower than that of qPCR within the same detection time. The system also demonstrated 100 % specificity and good precision (C.V. < 5 %) in detecting the three targets of the VP, providing a new approach for point-of-care testing (POCT) of VP.

Cellular and transcriptomic response to pathogenic and non-pathogenic Vibrio parahaemolyticus strains causing acute hepatopancreatic necrosis disease (AHPND) in Litopenaeus vannamei

The shrimp industry has historically been affected by viral and bacterial diseases. One of the most recent emerging diseases is Acute Hepatopancreatic Necrosis Disease (AHPND), which causes severe mortality. Despite its significance to sanitation and economics, little is known about the molecular response of shrimp to this disease. Here, we present the cellular and transcriptomic responses of Litopenaeus vannamei exposed to two Vibrio parahaemolyticus strains for 98 h, wherein one is non-pathogenic (VpN) and the other causes AHPND (VpP). Exposure to the VpN strain resulted in minor alterations in hepatopancreas morphology, including reductions in the size of R and B cells and detachments of small epithelial cells from 72 h onwards. On the other hand, exposure to the VpP strain is characterized by acute detachment of epithelial cells from the hepatopancreatic tubules and infiltration of hemocytes in the inter-tubular spaces. At the end of exposure, RNA-Seq analysis revealed functional enrichment in biological processes, such as the toll3 receptor signaling pathway, apoptotic processes, and production of molecular mediators involved in the inflammatory response of shrimp exposed to VpN treatment. The biological processes identified in the VpP treatment include superoxide anion metabolism, innate immune response, antimicrobial humoral response, and toll3 receptor signaling pathway. Furthermore, KEGG enrichment analysis revealed metabolic pathways associated with survival, cell adhesion, and reactive oxygen species, among others, for shrimp exposed to VpP. Our study proves the differential immune responses to two strains of V. parahaemolyticus, one pathogenic and the other nonpathogenic, enlarges our knowledge on the evolution of AHPND in L. vannamei, and uncovers unique perspectives on establishing genomic resources that may function as a groundwork for detecting probable molecular markers linked to the immune system in shrimp.

In vitro and in vivo evaluation of the efficacies of commercial probiotics and disinfectant against acute hepatopancreatic necrosis disease and luminescent vibriosis in Litopenaeus vannamei

The bioactivities of two commercially available probiotics and one chemical disinfectant were tested against strains of Vibrio parahaemolyticus (VPAHPND) and V. harveyi. This study aimed to determine shrimp pathogenic Vibrios' in vitro and in vivo sensitivities to commercial probiotics and a chemical disinfectant. The probiotics and disinfectant were tested first in vitro, followed by the in vivo trials. Results showed that upon administration of probiotics either through diet or adding into the tank water, the survivability of shrimp was increased during challenge with VPAHPND and V. harveyi. Also, the disinfectant was tested against the same pathogens and showed positive bactericidal effects at 2500 ppm and 5000 ppm. The present findings suggest that adding probiotics to the rearing water or the shrimp feeds effectively prevents infection by lowering the load of pathogenic bacteria. In comparison, the effectiveness of the disinfectant (PUR) depends on its appropriate concentration and timing of application. It is not only limited to rearing water but is also applicable for decontaminating pond liners, tanks, and other paraphernalia.

Presence of Zonula Occludens Toxin-Coding Genes among Vibrio parahaemolyticus Isolates of Clinical and Environmental Origin.

In recent studies, emphasis has been placed on the zonula occludens toxin (Zot) from the non-toxigenic Vibrio parahaemolyticus strain PMC53.7 as an agent inducing alterations in the actin cytoskeleton of infected Caco-2 cells and which appears as a relevant virulence factor. Universal zot primers were designed by the alignment of different types of zot gene and identification of conserved sequences to investigate the presence in diverse environmental and clinical V. parahaemolyticus isolates, in co-occurrence with virulence factors, such a hemolysins and secretion systems. The study screened a total of 390 isolates from environmental sources from Chile and Italy and 95 Chilean clinical isolates. The results revealed that around 37.2% of Chilean environmental strains and 25.9% of Italian strains, and 24.2% of clinical isolates carried the zot gene. The Zot-C2 cluster was present in 71.4% of Chilean environmental strains but absent in clinical isolates, while the Zot-C4 cluster was identified in 28.6% of environmental and 100% of clinical isolates. Understanding the role of zot in V. parahaemolyticus virulence is crucial, especially considering the risk associated with consuming diverse isolates from bivalves and the co-occurrence with virulence factors such as TDH, TRH or T3SS2.

Species Richness and Diversity of Gut Microbiota may Reduce AHPND in the Whiteleg Shrimp, Litopenaeus vannamei

Acute hepatopancreatic necrosis disease (AHPND) causes massive economic loss in shrimp culture sector. Eco-friendly feed additives, which can reduce antibiotic resistance in bacteria and antibiotic residues present in aquaculture products, have become popular in shrimp farming. These additives have proven to maintain a balance host gut microbiota in preventing pathogen infection. In the present study, gut microbiota of Litopenaeus vannamei, which had been administrated with different diets (probiotic: Bacillus B2; prebiotic: Gracilaria changii, G; and a combination of Bacillus B2 and G. changii, B2+G) and post-challenged with AHPND-causing Vibrio parahaemolyticus strain 3HP, was determined using the PCR-DGGE method. The results showed that the gut microbiota of L. vannamei was dominated by the phylum Proteobacteria. The abundance of Proteobacteria was higher in the B2+G and G treatment groups. High abundance of native gut microbiota, such as Proteobacteria might prevent the adhesion of virulent bacteria by competitive exclusion, and thus, reducing the chance of AHPND infection. Moreover, the shrimp from the B2+G treatment group with higher survival in the challenge tests possessed a higher diversity and species richness of gut microbiota. This suggests that higher diversity and species richness of beneficial gut microbiota probably interfering bacterial communication among the pathogens by either destructing or neutralising their quorum sensing signals. Consequently, this will prevent the colonisation of pathogens in the gut and resulted in a higher survival of the shrimp challenged with V. parahaemolyticus.

Serine protease inhibitor 3 (Serpin3) from Penaeus vannamei selectively interacts with Vibrio parahaemolyticus PirAvp.

Acute Hepatopancreatic Necrosis Disease (AHPND) represents a significant challenge in the field of shrimp aquaculture. This disease is primarily caused by Vibrio parahaemolyticus strains harbouring the pVA1 plasmid encoding the PirAvp and PirBvp toxins. To combat this epidemic and mitigate its devastating consequences, it is crucial to identify and characterize the receptors responsible for the binding of these pathogenic toxins. Our studied discovered that Penaeus vannamei's Serine protease inhibitor 3 (PvSerpin3) derived from shrimp hepatopancreatic tissues could bind to recombinant PirAvp , confirming its role as a novel PirAvp -binding protein (PA BP). Through comprehensive computational methods, we revealed two truncated PirAvp -binding proteins derived from PvSerpin3 called Serpin3(13) and Serpin3(22), which had higher affinity to PirAvp than the full-length PvSerpin3. The PA BP genes were amplified from a cDNA library that was reversed from total RNA extracted from shrimp, cloned and expressed in Escherichia coli. Three PA BP inclusion bodies were refolded to obtain the soluble form, and the recovery efficacy was found to be 100% for Serpin3 and Serpin3(13), while Serpin3(22) had a recovery efficacy of roundly 50%. Co-Immunoprecipitation (co-IP) and dot blot assays substantiated the interaction of these recombinant PA BPs with both recombinant PirAvp and VPAHPND (XN89)-producing natural toxins.

Genomic mining of Vibrio parahaemolyticus highlights prevalence of antimicrobial resistance genes and new genetic markers associated with AHPND and tdh + /trh + genotypes

BackgroundAcute Hepatopancreatic Necrosis Disease (AHPND) causes significant mortality in shrimp aquaculture. The infection is primarily instigated by Vibrio parahaemolyticus (Vp) strains carrying a plasmid encoding the binary toxin PirAB. Yet, comprehension of supplementary virulence factors associated with this relatively recent disease remains limited. Furthermore, the same holds for gastroenteritis in humans caused by other Vp genotypes. Additionally, given the prevalent use of antibiotics to combat bacterial infections, it becomes imperative to illuminate the presence of antimicrobial resistance genes within these bacteria.ResultsA subsampled number of 1,036 Vp genomes was screened for the presence of antimicrobial resistance genes, revealing an average prevalence of 5 ± 2 (SD) genes. Additional phenotypic antimicrobial susceptibility testing of three Vp strains (M0904, TW01, and PV1) sequenced in this study demonstrated resistance to ampicillin by all tested strains. Additionally, Vp M0904 showed multidrug resistance (against ampicillin, tetracycline, and trimethoprim-sulfamethoxazole). With a focus on AHPND, a screening of all Vibrio spp. for the presence of pirA and/or pirB indicates an estimated prevalence of 0.6%, including four V. campbellii, four V. owensii, and a Vibrio sp. next to Vp. Their pirAB-encoding plasmids exhibited a highly conserved backbone, with variations primarily in the region of the Tn3 family transposase. Furthermore, an assessment of the subsampled Vp genomes for the presence of known virulence factors showed a correlation between the presence of the Type 3 Secretion System 2 and tdh, while the presence of the Type 6 Secretion System 1 was clade dependent. Furthermore, a genome-wide association study (GWAS) unveiled (new) genes associated with pirA, pirB, tdh, and trh genotypes. Notable associations with the pirAB genotype included outer membrane proteins, immunoglobulin-like domain containing proteins, and toxin-antitoxin systems. For the tdh + /trh + genotypes (containing tdh, trh, or both genes), associations were found with T3SS2 genes, urease-related genes and nickel-transport system genes, and genes involved in a ‘minimal’ type I-F CRISPR mechanism.ConclusionsThis study highlights the prevalence of antimicrobial resistance and virulence genes in Vp, identifying novel genetic markers associated with AHPND and tdh + /trh + genotypes. These findings contribute valuable insights into the genomic basis of these genotypes, with implications for shrimp aquaculture and food safety.

Whole genome sequencing of nine Vibrio parahaemolyticus strains encoding (Pir) toxin-like genes from shrimp cultures in northern Peru using Oxford Nanopore technology.

Nine Peruvian isolates of Vibrio parahaemolyticus were characterized through sequencing, revealing the presence of simple sequence repeat, Pir toxin-like genes, and genes associated with antibiotic resistance, toxic components, and transposable elements. These findings expand our understanding of the genetic diversity, disease resistance, and virulence in cultivated shrimp populations in Peru.

Prevalence investigation of translucent post-larvae disease (TPD) in China

In early 2020, the shrimp farming in China suffered a severe setback due to an outbreak of the translucent post-larvae disease (TPD) in Penaeus vannamei. The emerging disease was caused by a hypervirulent strain of Vibrio parahaemolyticus (VpTPD). Although some progress has been made in understanding virulence factors of VpTPD, the diversity and prevalence of TPD pathogen are still unclear. Therefore, this study was undertaken to systematically investigate the epidemiology of TPD in China. In this study, potentially pathogenic bacteria present in naturally infected shrimp samples were isolated, identified, and preserved firstly. The further investigation showed that different Vibrio species carrying the key virulence genes of VpTPD can infect post-larvae of P. vannamei and cause TPD, revealing the diversity of TPD pathogens, and the pathogenic Vibrio strains has been temporarily named as Vibrio causing TPD (VTPD). In addition, epidemiological surveillance based on TaqMan qPCR detection and histopathology techniques analysis showed that VTPD positive samples were detected in most shrimp aquaculture ponds in different shrimp aquaculture provinces of China, with prevalence rate exceeding 50% in some areas. And seawater cultured species such as P. vannamei and P. japonicus were more sensitive to VTPD and were more likely to be infected or carry it. Freshwater cultured shrimp such as Macrobrachium rosenbergii and Procambarus clarkii may not be affected by VTPD. However, shrimp bait organisms are still at risk of carrying or being infected with VTPD. The result suggests that VTPD is still prevalent and spreading in coastal shrimp farming areas in China, with a high risk of transmission. This effort of present study provided a basic epidemiological understanding as well as important technical insights for prevention and management of emerging shrimp diseases.

Dietary Bromelain From Pineapple Extracts Enhanced Growth Performance and Modulated Stomach Microbiota Which Provided Longer‐Term Protection to Penaeus vannamei Against Acute Hepatopancreatic Necrosis Disease (AHPND) Caused by Vibrio parahaemolyticus

The emergence of acute hepatopancreatic necrosis disease (AHPND) caused by pathogenic strains of Vibrio parahaemolyticus (VPAHPND) had economically impacted shrimp farmers. There is great interest in using phytogenics as antibiotic alternatives in mitigating bacterial diseases. A 28‐day feeding trial using quadruplicate groups of Penaeus vannamei was conducted to evaluate the efficacy of bromelain (BM), a pineapple extract, on growth, gut microbiota, and resistance to AHPND. Dietary 1% or 2% BM enhanced growth of shrimp, which was significantly (P &lt; 0.05) higher at 2% BM compared to control (0% BM). In the first AHPND challenge, shrimp survival (mean ± SE) fed 1% or 2% BM were 84.8% ± 3.8% and 78.3% ± 2.5%, respectively, and significantly higher compared to the positive control group (69.6% ± 1.8%). Surviving shrimp were then regrouped into triplicates and fed the control diet for 14 days. This wash‐out period was used to determine if prior dietary BM had a longer‐term effect on shrimp health. In the second AHPND challenge, shrimp survival previously fed 1% or 2% BM was 71.1% ± 9.7% and 73.3% ± 3.8%, respectively, and significantly higher compared to the control group (51.1% ± 2.2%). Hepatopancreas showed less damage and harbored significantly lower Vibrio counts in shrimp fed BM‐added diets. Dietary BM modulated the stomach bacterial community and imparted the highest alpha diversity. At the phylum level, Proteobacteria, Actinobacteria, and Bacteroidetes were the main groups across treatments but with varying relative abundance. Luteolibacter, Paracoccus, Planctomyces, and Demequina were identified as the main contributors for the diversity differences observed among treatment groups. The relative abundance of Luteolibacter was significantly enriched and Vibrio bacteria significantly lowered in the stomach of the BM‐added groups. It was concluded that dietary BM induced modulation of the stomach microbiota of shrimp, which potentially enhanced its resistance to AHPND‐causing VP.

The impact of global warming on the signature virulence gene, thermolabile hemolysin, of Vibrio parahaemolyticus.

Global warming is increasing human exposure to pathogens and has already had an impact on human health. Vibrio parahaemolyticus, a major pathogen causing foodborne illness, accumulates in numerous aquatic organisms and can be affected by environmental stressors such as increased replication errors or DNA damage resulting in point mutations. This could lead to an elevation in the mutation rate, which influences the expansion of Vibrio spp. habitats and the spread of associated diseases. In this study, a total of 241 strains were isolated from aquatic products imported or exported through China Customs between 2005 and 2010. The whole genomes of those strains were sequenced, revealing a highly significant level of genetic diversity. Our analysis identified 27 new sequence types (STs) ranging from ST2950 to ST2976. The global temperature trend since 1950 affects the thermolabile hemolysin gene (tlh) found in all Vibrio parahaemolyticus leading to mutant sites exhibiting similar trends as temperatures rise; seven high-frequency mutation hotspots (A180G, T552G, G657T, T858C, C1062T, A1137G, and T1179C) were identified along with two clinically specific sites (T259C and A951T) that may indicate adaptation over time due to climate change, leading to increased virulence potential for this bacterial species. These results provide insight into the genetics of Vibrio parahaemolyticus and provide a reference for subsequent research, identification, and monitoring efforts related to its spread. IMPORTANCE In this study, Vibrio parahaemolyticus strains were collected from a large number of aquatic products globally and found that temperature has an impact on the virulence of these bacteria. As global temperatures rise, mutations in a gene marker called thermolabile hemolysin (tlh) also increase. This suggests that environmental isolates adapt to the warming environment and become more pathogenic. The findings can help in developing tools to analyze and monitor these bacteria as well as assess any link between climate change and vibrio-associated diseases, which could be used for forecasting outbreaks associated with them.

Bovine Lactoferrin and Hen Ovotransferrin Affect Virulence Factors of Acute Hepatopancreatic Necrosis Disease (AHPND)-Inducing Vibrio parahaemolyticus Strains.

Acute Hepatopancreatic Necrosis Disease (AHPND), a highly destructive shrimp disease, has inflicted severe setbacks on the shrimp farming industry worldwide. As the use of antibiotics is discouraged due to emerging antibiotic-resistant bacteria and the pollution of ecosystems, there is a pressing demand for novel, sustainable alternatives. Hence, the influence of bovine lactoferrin (bLF) and hen ovotransferrin (OT), two natural antimicrobial proteins, on the growth of three AHPND-causing Vibrio parahaemolyticus (Vp) strains (M0904, TW01 and PV1) was examined. Additionally, we explored their potential to affect selected Vp virulence factors such as biofilm formation, swimming and swarming, cell surface hydrophobicity, and activity of released lipases and caseinases. Lag phases of all bacterial growth curves were significantly prolonged in the presence of bLF or OT (1, 5 and 10 mg/mL), and bLF (5 and 10 mg/mL) completely inhibited growth of all strains. In addition, bLF or OT significantly reduced biofilm formation (all tested bLF and OT concentrations for Vp M0904 and Vp PV1), bacterial swimming motility (0.5 mg/mL bLF and OT for Vp M0904 and Vp TW01; 1 mg/mL bLF and OT for all strains), cell surface hydrophobicity (for all strains, all bLF and OT concentrations tested except for 0.125 mg/mL OT for Vp PV1) and lipase activity (1 mg/mL bLF and OT for all strains and 0.5 mg/mL bLF and OT for Vp PV1). These promising in vitro results suggest that bLF and/or OT might be used as novel agents for combating AHPND and warrant further research to elucidate the underlying mechanisms of action to fully unlock their potential for AHPND disease management.

125 Implementing a Genetic Evaluation Program for Acute Hepatopancreatic Necrosis Disease Resistance in Penaeus Vannamei (Pacific White Shrimp)

Abstract This study aims to evaluate the feasibility of a genomic evaluation program for Acute hepatopancreatic necrosis disease (AHPND) resistance in a commercial population of Penaeus vannamei. AHPND is caused by a strain of Vibrio parahaemolyticus (VpAHPND) producing a binary toxin. It is a World Organization for Animal Health (WOAH, Paris, France)-listed bacterial disease that has caused severe economic losses to the shrimp industry worldwide in recent years. The working hypothesis of this study is that the genetic variation in a commercial P. vannamei population allows an efficient genetic and genomic selection program. To investigate the genetic parameters for AHPND resistance, a controlled challenge experiment was performed at the Shrimp Improvement System biosafety laboratory in Miami, Florida, following an immersion challenge protocol using 4.95 × 107 CFU/ mL bacteria and Specific Pathogen Free (SPF) P. vannamei post larvae (n = 2,400) representing 40 selected families. The bioassay was terminated at 3 days post-challenge. Each family consisted of 60 animals, with one-half of the families (n = 20) representing a fast-growth (FG) line and the remaining one-half (n = 20) representing a slow-growth (SG) line. Survival was recorded as a binary of dead or alive at the end of the three days challenge experiment. Variance components were estimated under an animal threshold binary model using a Gibbs sampler. Estimation of genetic parameters was done within lines and by combining all data records. The average hazard risk ratio (HRR) of survival was 80% greater for the FG line (average mortality 40%) than the SG line (average mortality 20%, P &amp;lt; 0.05) based on survival analysis with a 31% mortality rate combining all data records. The posterior mean estimates of heritabilities were 0.14 (± 0.06) for FG, 0.16 (± 0.07) for SG, and 0.38 (± 0.08), combining all records. Estimated breeding values (EBVs) based on the animal threshold binary model ranged from -0.76 to 1.01 on the liability scale. Spearmans’ rank correlation coefficient between the average EBV of parents and the average mortality rates of corresponding families was 0.98. The results suggest that a breeding program for AHPND resistance is feasible. Moreover, the heritabilities suggest that both lines will respond to mass selection due to the presence of additive genetic variation for APHND-survivability. The next step of this project is to genotype animals from every family and implement a genomic selection program. Additionally, high throughput laboratory results and histopathology scores will be introduced in the genetic evaluation as correlated traits.

Change of antibiotic resistance in Vibrio spp. during shrimp culture in Shanghai

The judicious application of antibiotics in shrimp aquaculture is vital for addressing bacterial infections, yet monitoring bacterial resistance during distinct aquaculture phases remains deficient. This study focused on tracking antibiotic resistance dynamics in Vibrio strains sourced from both shrimp and their breeding settings. Over a standard aquaculture period (day 0, 20, 40, 60, and 80), we isolated eighty-eight Vibrio cholerae and ninety Vibrio parahaemolyticus strains from water, sediment, and shrimp samples. All isolates underwent assessment for antibiotic resistance phenotype and genotype. Both Vibrio species demonstrated resistance to nine antimicrobials. Notably, Vibrio cholerae isolates exhibited resistance rates of up to 46.6% for cephazolin, 30.7% for streptomycin, and 11.4% for ampicillin. Conversely, V. parahaemolyticus isolates displayed pronounced resistance to ampicillin (100%), cephazolin (62.2%), and cefoxitin (36.7%). Moreover, we identified 38 antibiotic resistance genes (ARGs) across all samples, with a higher prevalence observed in the water. Genetic diversity and clonal origins of the Vibrio isolates were quantified using Multilocus Sequence Typing (MLST). New Sequence Types (STs) were predominant among V. cholerae (95.4% of 43 isolates) and V. parahaemolyticus (92.9% of 28 isolates). Predominantly, V. parahaemolyticus isolates formed two extensive clonal groups: CC1420 and CC744. As the shrimp cultivation period progressed, both the resistance development rate among isolates and the average ARG count within the same STs of Vibrios steadily increased. This study underscores the potential role of ARGs transmission in the aquaculture environment as a crucial conduit for Vibrio antibiotic resistance development. Insights gained contribute to understanding the dissemination and transmission of antibiotic resistance in aquaculture, aiding in devising strategies to curb antibiotic resistance in aquatic environments.

Growth Performance and Disease Resistance against Vibrio parahaemolyticus of Whiteleg Shrimp (Litopenaeus vannamei) Fed Essential Oil Blend (Phyto AquaBiotic).

Acute Hepatopancreatic Necrosis Disease (AHPND) is a serious and emerging disease caused by a group of strains of Vibrio parahaemolyticus and affects farmed shrimp, particularly whiteleg shrimps (Liptopenaeus vannamei). The objective of this study is to assess the effect of dietary supplementation with two dosages of an essential oil mixture (Phyto AquaBiotic, abbreviated as PAB) on growth performance and mortality reduction after challenge against V. parahaemolyticus. PAB was mixed with basal diets at rates of 0, 1 and 2 g/kg and fed for 42 days. Each tank was stocked with 100 individuals with experimentation performed in triplicate. The results showed an improvement in growth performance in a dose-dependent manner, specifically regarding daily weight gain, specific growth rate and total biomass, which were significantly improved compared to control (p < 0.05). Further, PAB significantly reduced mortalities when challenged against Vibrio parahaemolyticus (p < 0.05) and decreased Vibrio spp. count in the hepatopancreas of infected shrimp. Overall, PAB was efficient in reducing mortalities in cases of disease outbreaks at a rate of 2 g/kg.

Terminalia catappa L. leaf extract interferes with biofilm formation of Vibrio parahaemolyticus and enhances immune response of Penaeus vannamei against acute hepatopancreatic necrosis disease (AHPND)

Crude ethanolic extract from Terminalia catappa L. leaves was investigated for its ability to interfere with biofilm formation of acute hepatopancreatic necrosis disease (AHPND)-causing and a reference Vibrio parahaemolyticus strains and modulate the immune response of Penaeus vannamei shrimp postlarvae. Results showed that biofilms formed by both AHPND-causing and reference strains of V. parahaemolyticus were rendered susceptible to dispersal upon treatment with the sublethal concentration of the extract. Furthermore, results of the qPCR analyses showed upregulation of six immune-related genes after 7 and 14 d of feeding. Pre-infection survival rates equivalent to the shrimp fed with un-supplemented feed suggest that T. catappa extract is safe for shrimp consumption. Upon infection with the AHPND strain, V. parahaemolyticus R1, the survival rate of the treatment group was 77% - significantly greater than those fed with un-supplemented feed. Similarly, those fed with T. catappa-supplemented feed and subsequently infected with V. parahaemolyticus ATCC17802 had a 93% survival rate, which is as high as the uninfected control group. These results ultimately indicate that feed supplementation with T. catappa increases the survival rate of the shrimp, possibly via attenuation of V. parahaemolyticus pathogenesis and stimulation of the shrimp's immune response against infections.