qPCR assay for detecting and quantifying a virulence plasmid in acute hepatopancreatic necrosis disease (AHPND) due to pathogenic Vibrio parahaemolyticus

Han, Jee Eun; Tang, Kathy F.J.; Pantoja, Carlos R.; White, Brenda L.; Lightner, Donald V.

doi:10.1016/j.aquaculture.2015.02.024

Cited by 80 publications

(51 citation statements)

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“…We infected Penaeus vannamei with Mexican (13-306/D4) and Vietnamese (13-028/A3) isolates in the laboratory bioassays, and 100% cumulative mortality was seen in both isolates by Day 3 (Han et al 2015b). Also, in other studies, we identified plasmids containing this Tn3-like transposon in 2 non-pathogenic V. parahaemolyticus isolates from Mexico (authors' unpubl.…”

Section: Discussionmentioning

confidence: 99%

Genotyping of virulence plasmid from Vibrio parahaemolyticus isolates causing acute hepatopancreatic necrosis disease in shrimp

Han

Tang²,

Lightner³

2015

Dis. Aquat. Org.

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Acute hepatopancreatic necrosis disease (AHPND) has caused severe mortalities in farmed penaeid shrimp throughout SE Asia and Mexico. The causative agent of AHPND is the marine bacterium Vibrio parahaemolyticus, which secretes PirA-and PirB-like binary toxin that caused deterioration in the hepatopancreas of infected shrimp. The genes responsible for the production of this toxin are located in a large plasmid residing within the bacterial cells. We analyzed the plasmid sequence from the whole genome sequences of AHPND-V. parahaemolyticus isolates and identified 2 regions that exhibit a clear geographical variation: a 4243-bp Tn3-like transposon and a 9-bp small sequence repeat (SSR). The Tn3-like transposon was only found in the isolates from Mexico and 2 unspecified Central American countries, but not in SE Asian isolates from China, Vietnam, and Thailand. We developed PCR methods to characterize AHPND-V. parahaemolyticus isolates as either Mexican-type or SE Asian-type based on the presence of the Tn3-like transposon. The SSR is found within the coding region of a hypothetical protein and has either 4, 5, or 6 repeat units. SSRs with 4 repeat units were found in isolates from Vietnam, China, and Thailand. SSRs with 5 repeat units were found in some Vietnamese isolates, and SSRs with 6 repeat units were only found in the Mexican isolates. KEY WORDS: Tn3-like transposon · Small sequence repeat · Genotying PCR · Shrimp disease · AHPND · Penaeus vannamei · Early Mortality Syndrome (EMS) Resale or republication not permitted without written consent of the publisherDis Aquat Org 115: [245][246][247][248][249][250][251] 2015 like toxin is the etiological factor for AHPND (G.C.F. Lo pers. comm.).In this study, based on published whole genome sequences of pathogenic V. parahaemolyticus strains, we analyzed the sequence variations within the virulence plasmid. We found 2 variable regions that correspond to the geographic collection sites of the isolates. We then developed and applied a duplex PCR assay that can serve to diagnose AHPND and, further, to distinguish among pathogenic strains collected from various geographic regions. MATERIALS AND METHODS AHPND Vibrio parahaemolyticus isolates and DNA isolationAHPND-V. parahaemolyticus isolates analyzed and used in this study are shown in Table 1. These include: (1) 9 whole genome sequences (WGSs) available in GenBank. These strains were collected from China, Vietnam, Thailand, and Mexico; (2) 26 bacterial isolates originally from Mexico and Vietnam. These bacterial isolates were sampled from pond water, sediments, and stomachs of shrimp affected by AHPND. Pure cultures were obtained by streaking on tryptic soy agar plus 2% NaCl (TSA+) plates and proved to be pathogenic by laboratory infection as described by Tran et al. (2013). Bacterial identifications were carried out using API Rapid NE test (bioMerieux Industry), 16S rRNA sequencing (Weisburg et al. 1991), and PCR targeting species (V. parahaemolyticus)-specific genes (toxR genes) (Kim et al. 1999). These b...

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Section: Discussionmentioning

confidence: 99%

Genotyping of virulence plasmid from Vibrio parahaemolyticus isolates causing acute hepatopancreatic necrosis disease in shrimp

Han

Tang²,

Lightner³

2015

Dis. Aquat. Org.

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“…Además, se han desarrollado métodos cuantitativos (qPCR), basados en una sonda TaqMan, con el fin de cuantificar los plásmidos presentes en V. parahaemolyticus, el cual genera un amplicón de 135 pb que se produce a partir de la secuencia de Pir A (Han et al, 2015c).…”

Section: Técnicas Moleculares Primers Utilizados Y Sus Particularidadesunclassified

Necrosis aguda del hepatopáncreas: una revisión de la enfermedad en Penaeus vannamei

Mejías

Peña²,

Aranguren

2017

Mesoam. Agron.

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“…Three V. parahaemolyticus strains were used as follows: EMS 13‐028/A3 (EMS) (Han, Tang, Pantoja, et al . ; Han, Tang, Tran et al . ), RIMD2210633 (RIMD) (Makino et al .…”

Section: Methodsmentioning

confidence: 99%

“…Histological examination of EMS‐infected shrimp has shown the disease causes tubule epithelial cells in the hepatopancreas to shrink and slough into the tubule lumen (Han, Tang, Pantoja, et al . ; Han, Tang, Tran et al . ).…”

Section: Introductionmentioning

confidence: 99%

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Strain and dose infectivity ofVibrio parahaemolyticus: the causative agent of early mortality syndrome in shrimp

et al. 2016

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Diseases of shrimp have contributed to billions of dollars of economic loss in the aquaculture industry. Newly emerging strains of the bacterium Vibrio parahaemolyticus produce a condition in shrimp called early mortality syndrome or acute hepatopancreatic necrosis disease. Three different V. parahaemolyticus strains were evaluated for their respective pathogenicity on shrimp, Litopenaeus vannamei, when the bacterial strains were grown under various laboratory conditions prior to inoculating shrimp. For each trial, feed was inoculated with a known concentration of bacteria and then fed to the shrimp. The early mortality syndrome strain of V. parahaemolyticus was the most lethal resulting up to 100% mortality within 24 h after being introduced to shrimp via a single feeding. The other two strains of Vibrio, one isolated from the environment and the other from a human clinical case, resulted in 0% and 30% mortality within 96 h respectively. The concentration of the early mortality syndrome strain of V. parahaemolyticus that the shrimp were exposed to directly correlated with mortality rate, which allowed for lethal or sublethal short‐term disease challenge assays to be established. Infiltration of haemocytes was also evident in the midgut caeca of shrimp infected with the early mortality syndrome strain of V. parahaemolyticus, which has not been previously reported.

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qPCR assay for detecting and quantifying a virulence plasmid in acute hepatopancreatic necrosis disease (AHPND) due to pathogenic Vibrio parahaemolyticus

Cited by 80 publications

References 9 publications

Genotyping of virulence plasmid from Vibrio parahaemolyticus isolates causing acute hepatopancreatic necrosis disease in shrimp

Genotyping of virulence plasmid from Vibrio parahaemolyticus isolates causing acute hepatopancreatic necrosis disease in shrimp

Necrosis aguda del hepatopáncreas: una revisión de la enfermedad en Penaeus vannamei

Strain and dose infectivity ofVibrio parahaemolyticus: the causative agent of early mortality syndrome in shrimp

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