Dissertation Final

DEVELOPMENT AND APPLICATION OF MANAGEMENT FOR FOUR SHRIMP DISEASES (TSV, YHV, WSSV AND NHP) IN THE WHITE SHRIMP Penaeus vannamei THROUGH DIFFERENT STRATEGIES

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Authors Aranguren, Luis Fernando

Publisher The University of Arizona.

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DEVELOPMENT AND APPLICATION OF MANAGEMENT FOR FOUR

SHRIMP DISEASES (TSV, YHV, WSSV AND NHP) IN THE WHITE SHRIMP

Penaeus vannamei THROUGH DIFFERENT STRATEGIES

Luis Fernando Aranguren Caro

A Dissertation Submitted to the Faculty of the

DEPARTMENT OF VETERINARY SCIENCE AND MICROBIOLOGY

In Partial Fulfillment of the Requirements For the Degree of

DOCTOR OF PHILOSOPHY WITH A MAJOR IN MICROBIOLOGY AND PATHOBIOLOGY

In the Graduate College

THE UNIVERSITY OF ARIZONA

2011

THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE

As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Luis Fernando Aranguren Caro entitled: Development and application of management for four shrimp diseases (TSV, YHV, WSSV and NHP) in the white shrimp Penaeus vannamei through different strategies and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy

______Date: September 29, 2011 Donald V. Lightner, Ph.D.

______Date: September 29, 2011 Beth Jacobs, Ph.D.

______Date: September 29, 2011 Kathy F.J Tang-Nelson, Ph.D.

______Date: September 29, 2011 David Besselsen, Ph.D.

______Date: September 29, 2011 Carlos Reggiardo, Ph.D.

Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.

______Date: September 29, 2011 Dissertation Director: Dr. Donald V. Lightner

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the copyright holder.

Luis Fernando Aranguren Caro

ACKNOWLEDGEMENTS

I would like to thanks the Colombian Corporation Center for Research in Aquaculture CENIACUA and The “Departamento Administrativo de Ciencia, Tecnología y Educación” COLCIENCIAS for providing me the scholarship award for my graduate studies at the University of Arizona.

I would like to thank my mentor and major advisor, Dr. Donald V. Lightner, for allowing me to become part of the Aquaculture Pathology Laboratory for these 5 years and for his endless patience, and for providing me valuable advice and feedback.

To my graduate committee members: Dr. Kathy Tang-Nelson, Dr. Beth Jacobs, Dr. Carlos Reggiardo and Dr. David Besselsen. Your guidance, teaching, and advice have been essential in completing this work.

I would like to thank to Dr. Chuck Sterling and Liz Gradillas in the Department of Veterinary Sciences and Microbiology for providing graduate teaching assistantships.

I would like to thanks Marcela Salazar for her guidance with my research. Special thanks are extended to lab members: Linda Nunan, Dr. Carlos Pantoja, Leone Mohney, Rita Redman, Solangel Navarro, Brenda Noble and Paul Schofield for their assistance, teaching and patience. I thank fellow graduate students, past and present: Thales Andrade, Lisbeth Echavarria and Allan Heres for their friendship and encouragement.

DEDICATION

To my parents: Augusto and Elsa de Aranguren for giving me their love and living experiences

To my wife: Yessenia, my sons Andrés and Lucas and my daughter Juliana, for being the inspiration every day

To my siblings Augusto and Monica for being more that a brother and a sister, my best friends

To shrimp

TABLE OF CONTENTS

LIST OF FIGURES ...... 8

LIST OF TABLES...... 10

ABSTRACT...... 11

CHAPTER 1. INTRODUCTION ...... 12 Literature review ...... 12 Description of etiological agents used in this research ...... 13 Strategies for diseases management ...... 16 Strategy for TSV management ...... 16 Strategy for YHV management ...... 18 Strategy for WSSV management ...... 19 Strategy for NHP management ...... 21

CHAPTER 2. CHARACTERIZATION OF A HIGHLY PATHOGENIC STRAIN OF TAURA SYNDROME VIRUS (TSV) FROM COLOMBIA SHRIMP CULTURE ...... 23 Abstract ...... 23 Introduction...... 24 Materials and Methods...... 27 Results...... 32 Discussion...... 38 Acknowledgments...... 43 Tables and Figures...... 44

CHAPTER 3. PROTECTION FROM YELLOW HEAD VIRUS (YHV) INFECTION IN Penaeus vannamei PRE-INFECTED WITH TAURA SYNDROME VIRUS (TSV) ...... 49 Abstract ...... 49 Introduction ...... 50 Materials and Methods ...... 53 Results ...... 57 Discussion ...... 59 Acknowledgments...... 63 Tables and Figures ……...... 65

TABLE OF CONTENTS - Continued

CHAPTER 4. USE OF A MARINE BACTERIUM (Vibrio sp.) AS A DELIVERY VEHICLE FOR WSSV VP28 GENE INTO SHRIMP Penaeus vannamei...... 69 Abstract...... 69 Introduction...... 70 Materials and Methods...... 73 Results...... 79 Discussion...... 81 Acknowledgments...... 84 Tables and Figures...... 85

CHAPTER 5. QUANTIFICATION OF THE BACTERIAL AGENT OF NECROTIZING HEPATOPANCREATITIS (NHP-B) BY REAL-TIME PCR AND COMPARISON OF SURVIVAL AND NHP LOAD OF TWO SHRIMP POPULATIONS ...... 91 Abstract...... 91 Introduction...... 92 Materials and Methods...... 94 Results...... 99 Discussion...... 103 Acknowledgments...... 108 Tables and Figures...... 109

REFERENCES………………………………………….………………………..….115

LIST OF FIGURES

Figure

1.1.... Colombian shrimp farming survival……….....……………………………...18

2.1 Neighbor-joining tree constructed from an alignment of the nucleotide

sequence of TSV full-genomes….…...……………………………………...46

2.2 Neighbor-joining phylogenetic tree from the alignment of the deduced

CP2 amino acid sequences among 59 TSV isolates. …………………...….47

2.3 Cumulative survival of Penaeus vannamei Kona stock infected with

three different TSV isolates: …….…...…..……………………………..…..48

3.1 YHV titration study in Penaues vannamei challenged by IM

injection with 4 different concentrations …….…...…………………………65

3.2 Cumulative survival of SPF Penaeus vannamei pre-exposed to

TSV that were challenged with YHV (1x104copies/shrimp) ..…………..….66

3.3 Comparison of YHV copy number in pleopods and LO of dead

shrimp exposed only to YHV vs. shrimp pre-exposed to TSV and

then challenged to YHV ………………..….....……………………….…...67

3.4 Histology of SPF Penaeus vannamei from infectivity studies with

TSV and then challenged with YHV …. ..………………………………….68

4.1 In vitro stability of the Vibrio 434 carrying the DNA plasmid

pGFP-uv-VP28 after about 554 generations….…...……………………..…87

LIST OF FIGURES - Continued

Figure

4.2 Detection of VP28 DNA at days 10, 15, 40, 50 post inoculation

in HP samples ………………………………………………………………88

4.3 In vitro translation of pGFPuv-VP28 in Vibrio 434. A:SDS-PAGE.

B: Western blot analysis of rVP28-GFP fusion protein in Vibrio 434….…..89

4.4 Challenge test of Penaeus vannamei immunized with Vibrio

434-pGFP-VP28 vaccine and challenged with 10 copies of WSSV by

intramuscular injection ……………………………………………………...90

5.1 Standard curve of the NHP-B copy number versus Cq

(Quantification cycle) value. Purified pNHP1………………………………112

5.2 Cumulative survival of COL and KONA Penaeus vannamei

populations challenged with NHP-B………………………………………. 113

5.3 NHP-B results using conventional NHP PCR.A: 25 cycles, B: 35 cycles….114

LIST OF TABLES

Table

2.1 Pair-wise comparison of nucleotides and amino acids sequences of TSV

CO 10 with six different full-length TSV isolates…………………………44

2.2 Mean ± standard error of TSV copy μg-1 of RNA in pleopods samples

of Penaeus vannamei Kona population challenged with three different

TSV isolates…………………………………………………………………45

4.1 Comparison of the Vibrio 434 16SrRNA sequence identity (%)

with top five sequences blasted in NCBI……………………………………. 85

4.2 Vibrio 434 colonization in hepatopancreas (HP) samples. Each value

is the mean of three replicates………………………………………………..86

5.1 Reproducibility of the TaqMan real time qPCR in six different assays.

SD: Standard Deviation; Cq: Quantification cycle……………………….…109

5.2 Final survival of two Penaeus vannamei populations infected with NHP-B

by reverse gavage…………………………………………………………...110

5.3 Mean ± standard error of NHP-B copy μg-1 of DNA in HP samples

of COL and KONA Penaeus vannamei shrimp populations in tanks 1

and 2 after NHP-B challenge test……………………………………….….111

ABSTRACT

A series of studies were conducted as part of my dissertation research on certain diseases of farmed penaeid shrimp and on strategies that might be applied to manage these diseases. These studies focused on the development and application of management for four shrimp diseases (TSV, YHV, WSSV and NHP) in the white shrimp Penaeus vannamei through different strategies. The studies focused on efforts to identify a new strain of Taura syndrome virus (TSV), and the prevention or mitigation of infection by

Yellow Head Disease (YHD), White Spot Disease (WSD), and Necrotizing

Hepatopancreatitis (NHP). The new strain of TSV reported in this study is among the most pathogenic strains discovered to date. Disease management strategies investigated include the prevention of YHD in the Americas by pre-exposing Specific-Pathogen-Free

(SPF) Penaeus vannamei to TSV. The other strategy investigated involved the use of a prototype “vaccine” that binds to the specific shrimp cell receptors and thus, prevents

WSSV from establishing an infection. The last strategy attempted to elucidate the reasons for the very low prevalence of NHP in commercial shrimp ponds in Colombia. It was found that through establishment of a breeding program in which shrimp were selected for resistance to TSV infection, Colombian shrimp farmers also, but indirectly, selected for resistance to NHP.

CHAPTER 1

INTRODUCTION

Literature review.

Shrimp diseases are among the main constraints to the sustainable growth of the shrimp industry. In 2010, according to the Office of International Epizootics (OIE), the diseases of penaeid shrimp that required official reporting by member country veterinary authorities were diseases caused by: White spot syndrome virus (WSSV), Taura syndrome virus (TSV), Infectious hypodermal and hematopoietic necrosis virus

(IHHNV), Infectious myonecrosis virus (IMNV), the complex of Yellow head viruses

(YHV/GAV) and the bacterial agent of necrotizing hepatopancreatitis (NHP-B) (O.I.E

2010). All of these diseases have caused estimated economic losses of more than 10 billion dollars to the worldwide shrimp farming industry (Lightner 2011).

This review describes the etiological agents used in the author’s research. All of these diseases are highly important pathogens that can affect the shrimp farming industry in the

Americas. This review is followed by a brief description of three strategies investigated for disease management. The first strategy was focused on TSV, and the effect of selective breeding for TSV resistance in the breeding program, conducted in Colombia, on the appearance of a new strain of TSV. The second strategy was aimed at providing an

13 explanation for the absence of YHD in the Americas and how enzootic TSV may be involved in this viral interference phenomenon. The third strategy was related to the use of a prototype “vaccine” employing a marine bacterium Vibrio sp. as a vehicle to deliver a recombinant protein that is critical for the pathogenesis of WSSV infection. Finally, the unexpected resistance of the Colombian shrimp populations to NHP was studied. It was determined that the resistance to NHP disease was acquired in parallel with resistance to

TS. This resistance explains the low prevalence of NHP in Colombian shrimp farming.

Description of etiological agents used in this research

Taura syndrome virus (TSV) is a non-enveloped, icosahedral virus that contains a single stranded positive-sense RNA genome of 10.2 kb (Bonami et al. 1997). This virus is a member of Dicistroviridae family (Mayo 2005). TSV contains two open reading frames

(ORFs). ORF1 encodes the non-structural proteins: helicase, protease and a RNA- dependent RNA-polymerase RdRp. ORF2 encodes three capsid proteins CP1 (40kDa),

CP2 (55 kDa) and CP3 (24 kDa) (Mari et al. 2002). The CP2 gene displays the greatest genetic variation and has been widely used to determine the genetic relationship among

TSV isolates (Tang and Lightner 2005; Wertheim et al. 2009).

Taura syndrome (TS), caused by TSV, emerged as a new disease in 1991 (Hasson et al.

1995; Brock et al. 1995; Lightner et al. 1995). TS was first reported in Ecuador in

Penaeus vannamei shrimp farms, and the virus was rapidly spread by the trade of infected postlarvae to most of the shrimp farming countries of the Americas, including

Colombia. In recent years, TSV has been reported in Asian, Middle Eastern and African countries such as Taiwan, Thailand, China, Indonesia, Saudi Arabia, and Eritrea in Africa

(Tu et al. 1999; Yu and Song 2000; Nielsen et al. 2005; Tang et al. 2005; Wertheim et al.

2009).

Yellow head virus (YHV) is an enveloped, positive sense, ssRNA genome of 26.6 kb that belongs to the genus Okavirus within the Roniviridae family (Cowley and Walker 2002;

Walker et al. 2005). YHV contains four ORFs. ORF 1 encodes for the protease, helicase, and RdRp. ORF 2 encodes for nucleocapside. ORF 3 encodes for the glycoprotein (gp

116 and gp64) and it is not known what ORF 4 encodes (Sittidilokratna et al. 2008).

Yellow head disease (YHD) causes high mortality in shrimp farms that reach up to 100%.

(Lightner 1996).

Yellow head disease (YHD) was first reported in Thailand in 1990 in Penaeus monodon juveniles (Limsuwan 1991; Boonyaratpalin et al. 1993). Subsequently, YHD was reported in other countries, including India, Indonesia, Malaysia, the Philippines, Sri

Lanka, Vietnam and Taiwan (Mohan et al. 1998; Wang & Chang 2000). YHV has been suspected to be present in cultured shrimp in the Western Hemisphere, and sporadic positive results by RT-PCR suggest the presence of this etiological agent. Nunan et al.

(1998b) and Durand et al. (2000) found YHV in imported frozen commodity shrimp from

Asia. In 1999 a sample from an Ecuadorian farm, showed a positive RT-PCR result for

YHV (Cenaim 2000). Recently, YHV was reported in the Pacific coast of Mexico in shrimp from several farms (De la Rosa-Velez et al. 2006).

White spot syndrome virus (WSSV) is a large dsDNA enveloped virus that belongs to the genus Whispovirus in the family Nimaviridae (Vlak et al. 2005). WSSV caused white spot disease (WSD). The WSSV genome has been completely sequenced and is approximately 300 Kb, with about 185 ORF (Yang et al. 2001; Van Hulten et al. 2001a).

To date, 50 structural proteins have been identified (Zhou et al. 2009), with four present in the viral envelope: VP 19, VP 24, VP 26 and VP 28 (Xie et al. 2006). Among these envelope proteins, VP 28 is the most abundant (Van Hulten et al. 2001b; Rout et al.

2007). Envelope proteins including VP 28 and VP 26 have been considered critical in the attachment and penetration of the virus into shrimp cells (Xie and Wang 2006; Yi et al

2004; Youtong et al. 2010).

WSD was first reported in China in 1992 and the disease rapidly spread to all of the

Asian countries that cultured P. monodon (Takahashi et al. 1994; Chou et al. 1995). In

1995, WSD was reported in the USA and its presence was linked to importation of frozen

WSSV infected shrimp from Asia (Nunan et al. 1998b; Durand et al. 2000) By 1998,

WSD (and WSSV) was present in most Latin-American countries that raised P. vannamei, and the disease caused very significant losses to the shrimp farming industry.

White spot disease (WSD) causes high mortality in shrimp farms that reach up to 100%.

(Lightner 1996).

Necrotizing hepatopancreatitis (NHP) is a disease caused by an unclassified Gram negative, pleomorphic intracellular alpha proteobacteria that will be called NHP-B for the purpose of this paper (Frelier et al. 1992; Lightner and Redman 1994). NHP affects cultured penaeid shrimp in several countries in the Western Hemisphere including Texas in the USA, most Central American and all South American countries that farm shrimp

(Lightner 1996). NHP was recently reported in Eritrea (Lightner et al. 2010). NHP is a chronic disease that causes mortalities up to 95% in shrimp populations in grow-out ponds (Lightner 1996; Johnson 1990) and broodstock ponds (Aranguren et al. 2006.

Morales et al. 2006). The occurrence of NHP is related to specific environmental conditions such as high temperature and high salinity (Lightner 1996; Vincent & Lotz,

2007). NHP-B-infected shrimp typically display a soft shell, flaccid bodies, lethargy, reduced feed intake and empty midgut (Lightner 1996). Hepatopancreas (HP) lesions in the acute phase of the disease include intense intercellular hemocytic response, a few to many melanized HP tubules with necrosis and sloughing off of HP tubule epithelial cells.

HP lesions in chronic phase include marked atrophy of the HP and its tubules, reduced epithelial cell height, low lipid storage and intratubular edema (Lightner 1996).

STRATEGIES FOR DISEASE MANAGEMENT

Strategy for TSV management

In Colombia, during the first years after the introduction of TSV, the disease caused high mortalities in shrimp farms that reached up to 100%. In an attempt to control the

17 epidemic, survivors from infected ponds were collected and used to initiate a mass selection-breeding program. This was later transformed into a family and within-family selection scheme, which incorporated TSV resistant animals into the breeding population.

This breeding program resulted in an increase in pond survival at harvest equal to levels prior to the initial TS outbreak (Cock et al. 2009) (Figure 1.1).

From 1999 through 2004, TS seemed to disappear from Colombian shrimp farms suggesting success of the breeding program where 100 % of the animals stocked in ponds were TS-Specific pathogen resistant (TS-SPR). During those years, almost no cases of TS were observed. The only way to demonstrate the presence of TSV in the farmed shrimp at that time was through the finding of spheroids in the lymphoid organ (a sign of chronic

TSV infection) by histological analysis.

In 2004, TS re-appeared in some ponds at about one month after stocking. The presence of birds and symptomatic shrimp in affected ponds that displayed the typical clinical signs of acute phase TSV infection was evidenced. Histological analysis showed typical acute phase lesions, which were characterized by the presence of severe necrosis and nuclear pyknosis of the cuticular epithelial cells and sub cutis and the typical “buck shot lesions” in cuticular epithelium of the stomach, gills and pleopods (Hasson et al. 1995).

-.( -.( 90.0 )*+,-.( /&'(0$%1+(-.( 80.0 !"#$%##"&'(

70.0

60.0

50.0

40.0 Survival(%)

30.0

20.0

10.0

0.0 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010

years FUENTE: SIC (Sistema Información CENIACUA)- "SICAM"(INVEMAR-CENIACUA)

Fig.1.1. Colombian shrimp farming survival in the Atlantic Coast between 1989 and 2010 (source: SIC Sistema informacion Ceniacua).

The hypothesis of the study presented in the Chapter 2, was to determine if the reappearance of TS in Colombian shrimp farms was due to a new strain of TSV and, if so, to determine its pathogenicity and its phylogenetic relationship with the known TSV clusters.

Strategy for YHV management

One of the common characteristics of YHV and TSV is the fact that they share many of the same target tissues. During acute phase, TSV and YHV infect the cuticular epithelium

(CE) of foregut and hindgut, stomach, gills and pleopods. Unlike TSV, YHV during the

19 acute phase targets also other organs such as the lymphoid organ (LO), connective tissue and hemocytes (Boonyaratpalin et al. 1993; Lightner 1996). During the chronic phase of infection, TSV and YHV produce LO spheroids in infected shrimp (Hasson et al.

1999a,b; Anantasomboon et al. 2008).

TSV and YHV share similar target tissues. Due to the fact that TSV was already present in the Americas when YHV was becoming pandemic in Asia, it is possible that TSV prevented shrimp from becoming infected with YHV. During this time frame, WSSV was spread to the Western Hemisphere through reprocessing of WSSV infected frozen material from Asia in shrimp packing plants located at coastal sites. Frozen product was demonstrated to also contain infectious YHV, but for some reason this virus did not become established in the Americas (Nunan et al. 1998b). Frozen commodity product infected with YHV and WSSV only resulted in the introduction of WSD to shrimp farms in the Western Hemisphere. Thus, the hypothesis of the work presented in the Chapter 3, was to evaluate if the viral interaction in shrimp pre-infected with TSV and then challenged with YHV under experimental conditions, protected the shrimp from YHD.

Strategy for WSSV management

After several attempts to develop a WSSV-resistant line through selective breeding programs with inconsistent results, some other alternative strategies to prevent and control WSSV have been proposed. The use of “vaccination” as a strategy to protect shrimp from White spot disease (WSD), has been recently reported with some promising

20 results. The most common target proteins used to prevent shrimp from WSD are the envelope proteins, principally VP28 and VP19. A short-term protection was afforded in

P. vannamei injected intramuscularly with VP 28 DNA and then challenged with WSSV

(Rout et al. 2007; Rajesh et al 2008, Li et al. 2009). Also, the purified VP28 protein has been employed through intramuscular injection into the shrimp that were then challenged with WSSV. The results indicated partial protection to infection by the virus (Rout et al.

2007; Wei and Xu 2009). The “vaccination” process appears to prevent the infection by blocking the virus from attaching to the shrimp cell receptors instead of activating the shrimp immune response. Although some of these experiments have shown protection in laboratory studies, unfortunately delivery of the “vaccine” requires injection of a DNA construct or recombinant protein which is not a feasible method in pond culture where millions of shrimp must be “vaccinated” and “re-vaccinated” at regular intervals during a typical grow-out cycle.

Recently, the use of attenuated bacteria has been tested as a potential delivery system for

DNA vaccines. Bacteria such as Escherichia coli (Witteveldt et al. 2004; Wei and Xu

2009), Bacillus subtilis (Fu et al. 2008) and Salmonella typhimurium (Ning et al. 2009) have been used as delivery vehicles that carry the gene for VP28 as a DNA “vaccine” In laboratory trials injection of these DNA “vaccines” into the shrimp provided them with protection from WSD. The limitation of using this type of bacteria is the fact that they are all fresh water bacteria that cannot survive or grow as normal micro flora associated with shrimp.

Vibrios are Gram-negative bacteria, usually present in high concentrations in estuarine and marine environments worldwide (Thompson et al. 2004). Vibrios are part of the normal bacterial community associated with shrimp, especially in the hepatopancreas

(HP) and gut, in some cases composing up to 85% of total bacteria present (Moss et al.

2000). Even though a few species have been reported as shrimp pathogens, most species are only opportunistic pathogens of shrimp and part of their normal micro flora (Lightner

1996). Various studies have shown that some Vibrio species can be used as probiotic bacteria that in high concentrations compete and colonize more efficiently than the pathogenic bacteria in the shrimp digestive tract (Gullian et al. 2004; Gomez-Gil et al.

2002; Vandenberghe et al. 1999).

The hypothesis for this study presented in the Chapter 4 was to use a non-pathogenic marine Vibrio sp., that are adapted to the same environmental conditions of salinity and temperature as cultured shrimp, as a vehicle to deliver a DNA “vaccine” into the shrimp’s digestive system in order to colonize it and continuously produce the recombinant protein

VP28, which appears promising as a blocking agent for prevention against WSSV infection.

Strategy for NHP management

22 infection have been restricted to ponds or tanks in which broodstock are raised and matured (Briñez et al. 2003, Aranguren et al. 2006). It is noteworthy that Colombian selection at the level of multiplication of broodstock is carried out under conditions where diseases such as Vibrio spp., IHHNV and others that are common in commercial grow out, are not excluded.

The hypothesis for this study, presented in the Chapter 5, was to evaluate the NHP-B resistance of shrimp from the TSV-genetic breeding program established in Colombia, and compares their resistance, to the disease using an SPF Kona line shrimp population for comparison. A final objective was to develop a qPCR protocol for the detection and quantification of NHP-B.

CHAPTER 2

CHARACTERIZATION OF A HIGHLY PATHOGENIC STRAIN OF TAURA

SYNDROME VIRUS (TSV) FROM COLOMBIA SHRIMP CULTURE

Luis Fernando Arangurena,b*, Marcela Salazarb, Kathy FJ Tanga, Donald V Lightnera

a. The University of Arizona, Aquaculture Pathology Laboratory, Dept. Veterinary Science and Microbiology, 1117 E. Lowell St., Tucson, AZ 85719, USA b. Corporacion Centro de Investigacion de la Acuacultura de Colombia, CENIACUA, Bogota, Cra 9B-113-60

*Corresponding author

E-mail address: [email protected], [email protected]

Abstract: Prior to 2004, Colombian shrimp farming benefited from a selection program in which Penaeus vannamei stocks were developed with resistance to Taura syndrome disease (TS). However since 2004, TS began to reappear as a significant disease. Pair- wise sequence analysis of the full-length TSV CO 10 genome was conducted and compared with six other isolates. This analysis revealed that the TSV CO 10 is closely related to other isolates that form the “Americas” group. Phylogenetic analysis using capsid protein 2 (CP2) region from different TSV isolates was performed. The analysis shows that the most recent TSV Colombian isolates (2006-2010) differ from the old

Colombian TSV isolates (1994-1998) by 4% nucleotide sequence and form a new

24 cluster. The relative pathogenicity of this new isolate (TSV CO 10) was determined through experimental infection of P. vannamei challenged with TSV CO 10, the reference isolate TSV Hawaiian (TSV HW) and most pathogenic isolate TSV Belize

(TSV BLZ). The results show similar cumulative survival and survival curves for the

TSV CO 10 and the TSV BLZ isolates. Viral quantification by RT-qPCR was carried out to determine the viral load in the TSV infected-shrimp exposed to each isolate. The TSV copy µgRNA-1 in the TSV-infected shrimp from Colombia and Belize was in the order of

1010, which was significantly higher (P<0.01) than that in shrimp infected with the TSV

HW isolate. This one log difference in viral load could explain the higher pathogenicity of the Belize and recent Colombian TSV isolates. This study suggests that the TSV present in Colombia shrimp farms during the last five years is a new TSV strain with increased pathogenicity.

Keywords

TSV, Dicistroviridae, Phylogenetic analysis, qPCR, Penaeus vannamei

Introduction

(ORFs). ORF1 encodes the non-structural proteins: helicase, protease and a RNA- dependent RNA-polymerase RdRp. ORF2 encodes three capsid proteins CP1 (40kDa),

CP2 (55 kDa) and CP3 (24 kDa) (Mari et al. 2002). The CP2 gene displays greater genetic variation and has been widely used to determine the genetic relationship among

TSV isolates (Tang and Lightner 2005; Wertheim et al. 2009).

Taura syndrome (TS), caused by TSV, emerged as a new disease in 1991 (Hasson et al.

1995; Brock et al. 1995; Lightner et al. 1995). TS was first reported in Ecuador in

Penaeus vannamei shrimp farms, and the virus was rapidly spread by the movement of infected postlarvae to most of the shrimp farming countries of the Americas including

Colombia. In recent years, TSV has been reported in Asian countries such as Taiwan,

Thailand, China, Indonesia, Saudi Arabia in middle East and Eritrea in Africa (Tu et al.

1999; Yu and Song 2000; Nielsen et al. 2005; Tang et al. 2005; Wertheim et al. 2009).

In Colombia, during the first years after the introduction of TSV, the disease caused high mortalities in shrimp farms that reached up to 100%. As an attempt to control the epidemic, survivors from infected ponds were collected and used to initiate a mass selection breeding scheme. This was later transformed into a family and within-family selection scheme, which incorporated resistant animals into the breeding population. This breeding program resulted in an increase of pond final survival equal to the levels to those prior to the initial TS outbreak (Cock et al. 2009).

Between 1999 through 2004, TS seemed to disappear from Colombian shrimp farms suggesting success of the breeding program where 100 % of the animals stocked in ponds

26 were TS-Specific pathogen resistant (TS-SPR). During those years, almost no cases of TS were observed. The only way to demonstrate the presence of TSV in the farmed shrimp at that time was through the findings of spheroids in the lymphoid organ by histological analysis.

In 2004, TS re-appeared in some ponds at about one month after stocking. The presence of birds and symptomatic shrimp in affected ponds that displayed the typical clinical signs of TSV acute phase infection was evidenced. Histological analysis showed typical acute phase lesions, which were characterized by the presence of severe necrosis and nuclear pyknosis of the cuticular epithelial cells and subcutis and the typical “buck shot lesions” in cuticular epithelium of the stomach, gills and pleopods (Hasson et al. 1995).

Several TSV strains have been identified based on phylogenetic studies and that allow for categorizing emerging TSV isolates into the known genotype clusters. Three clusters that group the genetic variants have been reported based on TSV CP2 sequence: Belize,

Americas, and SE Asia (Tang et al. 2005; Wertheim et al. 2009).

The objective of this study was to determine if the reappearance of TS in Colombian shrimp farms was due to a new strain of TSV and, if so, to determine its pathogenicity and the phylogenetic relationship with the known TSV clusters.

Materials and methods

TSV isolates and shrimp

The TSV isolates used in this study were from Hawaii in 1994 (HW; GenBank

AF277675), Belize 2001 (BLZ; GenBank AY590471), and Colombia 2010 (CO 10;

GenBank JF966384). All samples were stored at -70oC at the University of Arizona. The shrimp species used was Penaeus vannamei.

TSV and genomic sequencing

Frozen shrimp suspected of being infected with TSV from Colombia were shipped from

Colombia to the University of Arizona Aquaculture Pathology Laboratory (UAZ APL).

Total RNA was extracted from shrimp heads, excluding hepatopancreas, using the

RNeasy extraction kit (Qiagen) according to the manufacturer’s recommendations. The concentration of extracted RNA was determined by measuring the optical density (OD) at

260nm. RT-PCR was carried out according to Nunan et al. (1998a). For the sequencing of

Colombian TSV, primers amplifying 15 fragments of 1500 to 2000 bp were designed using Primer3 based on the TSV HW sequence. In order to amplify these long fragments, a Superscript III RT-PCR system with platinum Taq DNA polymerase was used. The cycling parameters consisted of 1 cycle at 45oC for 30 min, 94oC for 2 min, and 39 cycles at 94oC for 15 sec, 55oC for 30 sec and 68oC for 1.5 min with a final extension of 68oC for

7 min. PCR products were cleaned using QIAQuick PCR purification kit (Qiagen) and sequenced with an automated DNA sequencer ABI Prism 3730xl (Applied Biosystems).

Assembly of the sequence was carried out by overlapping amplified regions and using

TSV HW as a template using Geneious 4.8.5 software. The 10178 nt sequence of TSV

CO 10 is available in GenBank under the accession number JF966384.

The CP2 region of the different TSV isolates collected from Colombia (2006, 2007,

2010, years of collection), Mexico (2010) and Indonesia (2010) was amplified with the primers TSV 55 P1/P2 as described by Tang et al. (2005).

Distance and phylogenetic analysis

Nucleotide and amino acid sequence identity was determined using the BLAST utility from National Center for Biotechnology Information (NCBI). Nucleotide identity among the full-length genomes of the TSV isolates including, CO 10, HW, BLZ, Venezuela

2005 (VE 05, DQ212790), Thailand 2005 (TH 05, AY997025), China 2005 (CN 05,

DQ104696) and Texas, USA 2004 (TX-US 04, GQ502201), were analyzed using blastn.

Amino acid identities for the two ORF were analyzed using blastp: In ORF1: helicase, protease, RdRp and in the ORF2: CP1, CP2 and CP3. Pair-wise distance among amino acid sequences was estimated with the neighbor-joining (NJ) method using MEGA4

(Tamura et al. 2007).

Evolutionary relationship of the full-length genomes and the CP2 was analyzed. The sequence of CP2 was compared with 51 TSV isolates available in GenBank, and with six new CP2 sequences from Colombian TSV including 2006 (JN194141, JN194142 and

JN194143), 2007 ( JN194144 and JN194145), and 2010 ( JN194146), one CP2 from

Mexico 2010 ( JN194147) , and one from Indonesia 2010 ( JN194148) . The 3’ end of the CP2 was not complete in about 10% of the sequences, thus the analysis was restricted to 1150 nt (nt 7952-9101). The full nucleotide sequences were aligned using ClustalW

(Thompson et al. 1994). In addition, the translated CP2 coding region was aligned using

ClustalW. Phylogenetic analysis was conducted using MEGA4. A phylogenetic tree was calculated with the neighbor-joining (NJ) method (Tamura et al. 2007). The bootstrap consensus tree was inferred from 1000 replicates.

Quantitative SYBR Green RT-PCR

Primer express software (Applied Biosystems) was used to design the forward and reverse primers, TSV-306F (5’-CGT AAA TAG ACG GCC CAC AAA-3’), and TSV-

384R (5’ TGC ATC TAT ATA TCC AGG GAC TTA TCC-3’) from TSV genome to amplify a product of 79 bp upstream from ORF1. These primers matched 100% to the

CO 10, BLZ and HW TSV isolates in that region. Amplification was carried out in a final volume of 20 μl with 10 µl of SYBR-Green Master Mix (Applied Biosystems), 0.3 μM of each primer, 1 µl of 40X Multiscribe and RNase inhibitor mix and 1 µl of RNA. The RT- qPCR method consisted of 30 min at 48°C and 10 min at 95°C followed by 40 cycles of

15 s at 95 °C and 1 min at 60 °C. Amplification detection and data analysis were performed with the master cycler Realplex 2.0 (Eppendorf).

To generate an RNA standard for the RT-qPCR, primers TSV-285F (5’- TTC TAT AGG

TCT GGT TTA AAA CGT AAA 3’-) and TSV-516R (5’-CGG TTT TCT CCA TCA

TCG TT -3’) were used to generate a 232 bp amplicon. The PCR products were cleaned with a QIAquick PCR purification kit (Qiagen), ligated to a pGEM-T Easy vector

(Promega) and transformed into Escherichia coli JM109 cells (Promega). The recombinant plasmid pTSV, was verified by DNA sequencing using an automated DNA sequencer ABI Prism 3730xl (Applied Biosystems). The plasmid was then linearized by digestion with SpeI (New England Biolabs) and cleaned with a QIAquick PCR purification kit (Qiagen). The plasmid was used as a template for an in vitro transcription with MEGA scripts kit (Ambion) following the manufacturer’s protocol. 1 µl of TURBO

DNase was added to the mixture and incubated 15 min at 37oC. For the recovery of the

RNA, Ambion MEGAclear kit was used following the recommendation of the manufacturer. The synthesis of RNA was confirmed by electrophoresis 1.5% agarose gel containing 0.5 μg ml-1 ethidium bromide, visualized under ultraviolet light and digitally photographed by the AlphaImager (Alpha Innotech). To determine the yield, a reading at

o A260 was conducted in a spectrophotometer. RNA was stored at -70 C for further analysis.

Challenge test

To determine the pathogenicity of TSV CO 10, an experimental infection was carried out at the UAZ-APL. Three TSV isolates were used in this study: TSV CO 10 with an unknown pathogenicity, TSV BLZ (that is the most pathogenic TSV isolate) and TSV

HW was used as the reference isolate. Specific-Pathogen-Free (SPF) P. vannamei, Kona

31 line (Pruder et al. 1995) were used for these tests and obtained from the Oceanic Institute.

The average weight of the shrimp was 3.0 g.

TSV infected tissue was prepared using the three different TSV isolates. Only frozen

TSV-infected shrimp heads, excluding hepatopancreas, were homogenized in a buffer

(0.02 M Tris-HCl, pH 7.4, 0.4 M NaCl buffer; 1g/10 ml), using a tissue blender and clarified at 3500 x g for 20 min and 5000 x g for 20 min at 4oC. Samples were diluted

1:20 in 2% saline, aliquoted and frozen. Three tanks (one for each TSV isolate) were set up with 10 shrimp per tank. Shrimp were infected with a single injection (100 µL) of

TSV inoculum into the second abdominal segment. Moribund and dead shrimp were frozen and stored at -70oC for later use in the TSV challenge tests.

Total RNA was extracted from 30 mg of each TSV inoculum as described above. A RT- qPCR assay was conducted to quantify the viral content for each TSV inoculum. There were four experimental groups; one negative control and one for each TSV isolate (CO

10, BLZ and HW). Each group had three replicate 90 L tanks that contained ten 3.0 g shrimp each. The salinity and temperature was adjusted to 25 ppt and 27±1oC, respectively. Shrimp were infected with the different fresh TSV inoculum by a single injection of 2.5x106 TSV copies/shrimp in a 50 µL volume.

Mortality was recorded daily from the start of the experiment. Moribund shrimp were fixed in Davidson’s AFA fixative for histological analysis (Bell and Lightner 1988) or

32 immediately frozen for molecular analysis. Dead animals were frozen and stored at -70°C for RT-qPCR analysis. Ten shrimp from each TSV isolate challenge were used to determine the TSV viral load. Pleopods from the first abdominal segment were removed from each shrimp and about 25-30 mg of tissue was used for total RNA extraction. For the RT-qPCR reaction all the templates were adjusted at 20 ng of RNA/µl.

Statistical analysis

Statistical analyses were conducted in STATA IC10. The survival analysis and the cumulative survival probabilities were determined by Kaplan Meier survival analysis.

Bartlett test was used to determine the equal variances. One-way ANOVA (α=0.05) was used to determine the difference of viral load among groups after logarithmic transformation of data. Post-Hoc Bonferroni test was used to determine the significant groups.

Results

Sequence analysis

The full TSV CO 10 genome was sequenced and the 10178 nucleotide sequence was compared to six other TSV genomes including HW, BLZ, VE, TX-US 04, CN, and TH isolates. The nucleotide and the deduced amino acids identity of these genomes compared to the Colombian isolate are shown in Table 2.1.

Mean nucleotide identity of TSV CO 10 with six full-length TSV isolates was 95%. The

TSV CO 10 was most similar to the Hawaiian TSV (96%) and most distant to the

Venezuela TSV isolate (92%). The amino acid identities among the genes from the ORF1 were highest among all the TSV isolates, with helicase having the highest identity percentage (98.8%), followed by the protease (98.5%) and RdRp (98.0%). Interestingly there is a high identity in RdRp from the Colombian and Belize isolates (99%). When analyzing aa 49 of RdRp in all isolates, only the TSV CO 10 and BLZ have an isoleucine

(Ile) while all the other isolates have a leucine (Leu) at this position. At amino acids 65 and 191, there is a valine (Val) in TSV CO 10, in all others there is a methionine (Met).

At aa position 236, there is a aspartic acid (asp) in the Colombian TSV isolates and in all others there is a glutamine (Gln). Finally, at position aa 253, only the Colombian TSV isolates have a glycine (Gly), the Hawaiian and Venezuelan TSV isolates have a valine

(Val) while the others have a asparagine (Asn).

As expected, ORF2 was more variable than ORF 1, especially in the CP2 region (93%), where identity ranged from 90% in Venezuela to 95% in Hawaii; this result being in agreement with other studies (Mari et al. 1998; Tang et al. 2004; Wertheim et al. 2009;

Dhar et al. 2010;). The deduced amino acid sequence more conserved among all the TSV isolates was the CP3 with a 99.2% identity.

Phylogenetic analysis

Two types of phylogenetic analysis were conducted, by using the full-length genomic sequence and by using the deduced aa of the CP2 (Fig. 2.1).

The full-length genome phylogenetic tree described above, agrees with the recent phylogenetic study (Dhar et al. 2010). Even thought the identity percentage between

TSV CO 10 and TSV Venezuela (VE) is the lowest (92%), the closest position of the

TSV VE isolate to the TSV CO 10 in the phylogenetic tree, suggests that TSV CO 10 and

TSV VE were derived from the same ancestor. Also the close association between Texas and the Asian isolates that has been previously reported (Tang et al. 2005; Wertheim et al. 2009; Dhar et al. 2010), and hypothesized to be related to the movement of frozen shrimp for reprocessing at a plant near the affected shrimp farm (Dhar et al. 2010). The

CP2 phylogenetic tree of the deduced amino acid is shown in the Fig. 2.2

The deduced amino acids sequences of CP2 from 59 different isolates is in agreement with the three clusters (Americas, Asia, and Belize) described by Tang et al. (2005), although bootstrap values for all the clusters is lower than those reported in that study.

The Asian cluster shows a bootstrap value of 51%, Belize shows a bootstrap value of

99%, and the American cluster a bootstrap value of 43%. The mean distance between

Americas TSV and SE Asia was relatively low (3.5%), followed by the distance between

BLZ and SE Asia (3.9%) and BLZ and Americas (4.9%). The mean distances within each group were 1.1%, 1.2% and 3.0% for BLZ, SE Asia and Americas, respectively.

The high mean distances for CP2 sequence identities in the Americas TSV isolates is explained by the presence of three sub clusters derived from the Hawaiian TSV that is considered to be the original ancestor: Colombia and Venezuela in South America and a

Mexico sub cluster in Central America. The Colombian sub-cluster has a bootstrap value

100% indicating clearly that this group differs from the previous Colombian TSV isolates

(TSV CO) (1994-1998) (Fig. 2.2). This sub cluster group is for TSV isolates taken from

2006 through 2010. The Venezuela sub-cluster has a high bootstrap value (99%), being similar than the bootstrap value reported by Cote et al. (2008). The Mexico sub-cluster nest TSV isolates from Mexico 1998 to 2010 (including a sample from Eritrea 2004) with a bootstrap value of 90% that differs from the original TSV as well (Fig. 2.2). Based on the full-length genome phylogenetic tree, the common ancestor for all three sub-clusters is the Hawaiian TSV (Fig. 1). In this ancestral cluster, samples of the TSV HW CP2 data from 1994 through 2003 were unique relative to more recent isolates. None of the recent

TSV CP2 isolates have nested within this group. Unlike the original HW cluster, in which most of the shrimp populations exposed to TS were TSV susceptible, in the new three sub-clusters, shrimp populations exposed to TS are TS-SPR populations.

The mean pair-wise distance between TSV CO 10 and TSV CO was 4.0%, between TSV

CO 10 and TSV VE it was 5.6%, and between TSV CO and VE was 4.1%. These distances indicate that TSV CO 10 and TSV CO are the most closely related. The mean pair-wise distances within each group were 0.7%, 1.0 and 2.4% for TSV CO 10, TSV CO and TSV VE respectively.

When comparing the amino acid change among the 383 aa region from the TSV CP2, there were 73 substitutions in the 59 different isolates with most of them at the C- terminal region. When comparing amino acid sequences between TSV CO and TSV HW, there were just 9 aa substitutions, while between TSV CO 10 and TSV HW, there were

18 aa substitutions. The dissimilar changes in aa between TSV CO and TSV CO 10 were

14 at these aa positions: (66) Lys in the TSV CO for an Arg in the TSV CO 10, (106)

Val-Ile, (212) Ser-Pro, (217) Asn-Asp, (280) Glu-Lys, (287) Leu-Val,(288) Tyr-Ser,

(296) Ser-Lys, (297) Arg-Lys, (306) Pro-Ser, (359) Val-Ile, (364) Glu-Gly, (378) Glu-

Gly, (380) Thr-Ala.

Challenge test

Pathogenicity of TSV CO 10 was evaluated against TSV BLZ (the most pathogenic isolate) and with the TSV HW (the less pathogenic and reference isolate). Results are shown in Fig. 2.3.

Final survival in shrimp challenged with TSV CO 10 and TSV BLZ was 0% after 5 and 6 d.p.i, respectively, while survival in shrimp challenged with Hawaiian TSV was 3% after

8 days post infection (d.p.i). Even thought the general cumulative survival was very low, the survival curve of TSV HW was significantly different from TSV CO 10 and TSV

BLZ vs. TSV HW (p<0.001). The estimated time for 25% survival was 3 days in TSV

CO 10 and TSV BLZ isolates, compared to 5 days in the TSV HW population. In general, the final survival of the SPF line after challenge to TSV HW is about 20-30%

(Tang et al. 2004; Erickson et al. 2005, Srisuvan et al. 2006). A possible genetic changes in the different Kona SPF batches may be among the reasons for this increase in susceptibility.

Through histological analysis of moribund shrimp, the typical TSV acute phase was present in all three groups. Multifocal areas of necrosis of the cuticular epithelium of gills, appendages, foregut and hindgut were observed. Infected cells displayed nuclear pyknosis and karriorexis with cytoplasmic eosinophilia with the characteristic “buckshot lesions” pathognomonic for TSV (Lightner et al. 1995; Hasson et al. 1995). No presence of lymphoid organ spheroids was observed in any of the samples analyzed, nor was there evidence of any other disease present in the moribund shrimp analyzed.

Quantitative (q) RT-PCR

Ten samples from each of the three TSV isolate groups were analyzed by RT-qPCR.

TSV was present in all animals analyzed for all the isolates. TSV copy number in shrimp infected with BLZ and CO 10 TSV was significantly higher than in shrimp infected with

TSV HW (p<0.05) as shown in Table 2.2. There was no significant difference in the

TSV viral load between CO 10 and BLZ.

Discussion

New TSV isolate

In this study we compared the full-length genome sequences of seven TSV isolates. The

TSV CO 10 is grouped within the Americas TSV cluster (Fig. 2.1). Based on the phylogenetic tree, VE 05 and CO 10 TSV isolates come from the same ancestor. The

Asian isolates are also related among each other and Belize TSV still remains as a unique

TSV (Fig. 2.1). The diversity of TSV in CP2 was greater than previously reported by

Tang et al. (2005) where 17 American TSV CP2 isolates reported a bootstrap value of

62% and Cote et al. (2010) where 17 American TSV CP2 isolates (including Venezuelan

TSV) reported a bootstrap of 50%. In this study, with 34 American TSV CP2 isolates, the bootstrap value was 38%. This finding is explained in part by the three sub-clusters present in the Americas; the new TSV Colombian sub-cluster (2006-2010), the

Venezuelan sub-cluster (2005-2006) and the Mexican sub-cluster.

The challenge test shows clearly that CO 10 and TSV BLZ isolates were more pathogenic than the TSV reference isolate from Hawaii (p<0.001). In part the higher pathogenicity is explained by the higher viral load in BLZ and CO 10 in comparison with TSV HW

(Table 2). Tang et al. (2004) reported viral loads for TSV in the order of 5.8x108 TSV copies μg RNA-1 in SPF shrimp exposed to Hawaiian TSV. Srisuvan et al. (2006) compared SPF populations exposed to different TSV isolates including Belize, Hawaii,

Thailand and Venezuela. In that study, similar results were found regarding the viral load.

Belize TSV-infected shrimp had the highest viral load (1x107 μl RNA-1) and lowest survival (0%).

Among the seven full-length TSV isolates, The RdRp aa sequence between BLZ and CO

10 TSV isolates show the highest identity (99%). TSV form the basic fingers-palm- thumb domain structure similar to others polymerases (Dhar et al. 2010), thus, possible changes in tertiary structures due to point mutations in the RdRp could contribute to change the replication fidelity. Four mutations points were identified in TSV CO 10

RdRp that could be involved in changes in tertiary structure, enhancing the RdRp activity that could explain the higher viral load in comparison to the reference HW isolate. One aa change at position 49 (Ile) only found in CO 10 and BLZ could be involved in the increased efficiency of the TSV RdRp. Bull et al. (2010) found a point mutation in

Norovirus RdRp in one aa at position 291 from Lys to Thr or Val, increased the nucleotide incorporation rate to about 20.2%. Qin et al. (2001), found in the Hepatitis C virus a point mutation in the region involved in RNA template binding and RNA template/primer binding that was essential for RdRp activity.

TSV evolution

TSV has evolved relatively fast to the point that recents TSV CP2 do not nest with the original TSV from Hawaii (Fig.2.2). The last TSV CP2 isolate that grouped within the

Hawaiian isolate was from Honduras 2003. Robles-Sikisaka et al. (2002) reported that some TSV samples from Mexico and Nicaragua failed to react with the monoclonal

40 antibody 1A1(Mab 1A1) that react well with Hawaiian TSV. This Mab1A1 does not react with either Belize TSV (Erickson et al. 2005), Venezuela (Cote et al. 2009) or

Colombia TSV. This suggests that the original TSV ancestor no longer exists and that the virus has evolved and diversified. This TSV evolution is a consequence of a selection pressure and the higher number of mutations due to the lack of proofreading function in the RNA-dependent RNA polymerase that allows the genome to undergo a rapid mutation rate. Wertheim et al. (2009) reported the TSV nucleotide substitution rate at

2.37x10-3 substitution/site/year, which explain the high diversity in different TSV regions especially in the CP2.

The CP2 region from the new Colombian TSV isolates form a new cluster that differs from the TSV CO isolates from 1994 and 1998. This change has been reflected in changes in TSV pathogenicity and noticed at the farm level. From 1999 through 2004 the presence of TS in farms was not evident. During that time all the post larvae stocked in the farms were a (TS-SPR) line from the Colombian Corporation Center for Reseach in

Aquaculture (CENIACUA) breeding program. Hence, even though TSV was still present, TS-SPR did not display typical TSV acute phase pathology. Indication that TSV was still present during that time at the farm level, was the presence of lymphoid organ spheroids (indicating chronic phase TSV infections) present in broodstock that were bred in farm conditions and confirmed by in situ hybridization. After 2004, shrimp showing

TSV acute phase signs were detected in several farms. The presence of TSV was correlated with the lower survival results during those years. In 1999, the final survival

41 in the Atlantic shrimp farms was 65.5% increasing steadily up to 75.8% in 2004. During the next years after the re-appearance of TSV acute phase lesions (2004-2010), the final survival has decreased progressively to the point that in 2010 it was 58.7%. During those years (2004-2010), shrimp farming densities increased from 22 shrimp/m2 to 43 shrimp/m2 in 2010. The increase of stocking density is a factor that affects final survival in ponds and density could have been the “trigger” for the TSV CO 10 to appear in the

Colombian farms.

CP2 surface protein

The presence of acute TSV in the TS-SPR line, without causing massive mortalities at farm level, indicates that there is a partial protection of the TS-SPR line to this new TSV strain. Srisuvan et al. (2006) using HW, BLZ, Thailand and Venezuela TSV isolates, found different degrees of resistance of the TS-SPR shrimp selected against TSV HW. In the challenge against TSV BLZ, the final survival was 77% whereas with TSV HW it was 100%. Moss et al. (2005) found similar results in a TS-SPR line with a final survival of 63% vs. 93% using TSV BLZ and TSV HW respectively. These results confirm that even though there is some protection to another mutated TSV strain, the protection is not complete. An explanation could be related to changes of the CP2 that could change binding activity to the host cells. Senapin and Phongdara (2006); Busayarat et al. (2011) have suggested that laminin receptor is the shrimp cell receptor for TSV. This laminin specifically binds to TSV-CP2, suggesting that this protein is critical for the virus attachment to the shrimp cell. Poulos et al. (1999) and Cote et al. (2009) in the attempt to

42 produce Mab against all TSV strains, produced Mabs that react specifically with the

TSV-CP2 region, which suggests that this is the most antigenic region. Mab1A1 (Poulos et al. 1999) reacts only with TSV HW and the native form of TSV VE and Mab 2C4

(Cote et al. 2009) only reacts with HW, BLZ and Mexican TSV isolates. This suggests that mutations in CP2 gene can alter the CP2 protein configuration and can influence the affinity between the virus and the host cells receptor. It is known that a high-affinity receptor is required for virus entry (Strauss and Strauss, 2008). Thus, possible changes in aa composition in CP2 configuration can alter the affinity to the receptor either increasing or decreasing binding and therefore change the pathogenicity of the virus.

Changes in CP2 and RdRp in the TSV CO 10 are likely to be the result of “viral fitness” which depends on many factors including viral mutation, replication efficiency, population size and host factors. Viruses increase their fitness if they are able to produce more and more diverse populations (Montville, 2005). Increased replication rate would produce a larger heterogeneous population than a slower replicating virus (Bull et al.

2010).

In summary, we have described a new strain of TSV in Colombia that is highly pathogenic, in part due to the higher viral load and changes in CP2 configuration. The diversity of the TSV CP2 in the Americas is greater than before, because of the two new sub-clusters in South America (Venezuela and Colombia) and the new sub-cluster in

Mexico. The more TSV samples that are analyzed, the more likely it is that the source of a particular TSV epizootic can be inferred based on the homology of its CP2 gene.

The decrease of the final survival in Colombian shrimp farm culture is explained partially by the new TSV strain that is infecting the TS-SPR lines. This result, and the partial protection of TS-SPR lines when exposed to a mutated strain, creates the need for genetic programs to develop TS-SPR lines using a regional TSV strain that confers high specific protection to the shrimp farmed in a specific region.

Acknowledgments

This work was supported by the Gulf Coast Research Laboratory Consortium Marine

Shrimp Farming Program, USDA Grant No. USMSFD 2010-38805-21115. We thank to

Linda Nunan and Brenda Noble, Xenia caraballo, Boris Briñez for providing technical assistance and Rita Redman for histological services. We also want to thank Colciencias for providing funds for the sequencing of the CP2 (2006-2007) samples from Colombia.

Tables and Figures

Table 2.1 Pair-wise comparison of nucleotides and amino acids sequences of TSV CO

10 with six different full-length TSV isolates.

TSV Nucleotides Amino acid identity (%) isolates identity (%) ORF1 ORF2 Overall Helicase Protease RdRp CP1 CP2 CP3 Hawaii 96 99 99 98 98 95 100 Belize 95 99 98 99 98 94 99 Venezuela 92 99 97 97 97 90 97 Texas 95 98 99 98 97 93 99 China 95 99 99 98 97 93 100 Thailand 95 99 99 98 97 93 100 Mean 94.7 98.8 98.5 98.0 97.3 93.0 99.2

Table 2.2 Mean ± standard error of TSV copy μg-1 of RNA in pleopods samples of

Penaeus vannamei Kona population challenged with three different TSV isolates.

TSV strain N Mean Copies μg-1of RNA± (SE)

Hawaii 10 9.6 x 109 ± (1.4 x 1010)

Colombia 10 *1.7 x 1010 ± (8.8 x 109 )

Belize 10 * 2.8 x 1010 ± (2.5 x 1010)

* indicate significant differences (p < 0.05).

Fig. 2.1 Neighbor-joining tree constructed from an alignment of the nucleotide sequence of TSV full-genomes. Numbers indicate the bootstrap support from 1000 replicates. Scale bars shows the evolutionary distance of 0.005 nucleotides substitution per position.

Thailand 100 SE Asia TSV cluster 100 Texas China Hawaii

100 Venezuela 88 Colombia Belize Americas TSV cluster

Belize TSV cluster 0.005

Fig. 2.2 Neighbor-joining phylogenetic tree from the alignment of the deduced CP2 amino acid sequences among 59 TSV isolates. Numbers indicate the bootstrap support from 1000 replicates. Scale bars shows the evolutionary distance of 0.002 aa substitution per position. Asian and Belize TSV isolates are condensed.

MX 05 49 HW 07 64 MX 04 82 ER 04 MX 07 90 Mexican sub-cluster 54 MX 10 53 MX 98 27 US 96 35 MX 95B 41 MX 95A HW 95 52 US 98

19 HO 94 16 EC 93 Ancestor sub-cluster EC 94 HW 94 65 4 MX 95C HO 03 38 31 CO 94C CO 94A 87 CO 94D 71 CO 94B CO 98

58 CO 06B 37 54 CO 06A

100 CO 06C CO 07A Colombian sub-cluster 48 22 CO 10 35 CO 07B

55 EC 06A EC 06B 99 AW 05 Venezuela sub-cluster 32 VE 05A 28 VE 05B

Asian isolates (18) 49 Belize isolates (7) 99

0.002

Fig. 2.3 Cumulative survival of Penaeus vannamei Kona stock infected with three different TSV isolates: TSV HW (), BLZ (−−−), CO 10 ( ) and the control group ( ). Data represents the average of three separate replicates. Error bars represent the 95% confidence interval of the three data sets.

Hawaii Col Belize Control

100 90 80 70 60 50 40 30

CumulativeSurvival(%) 20 10 0 1 2 3 4 5 6 7 8 days post infection

CHAPTER 3

PROTECTION FROM YELLOW HEAD VIRUS (YHV) INFECTION IN Penaeus

vannamei PRE-INFECTED WITH TAURA SYNDROME VIRUS (TSV)

Fernando Arangurena,b*, Kathy FJ Tanga, Donald V Lightnera

a. The University of Arizona, Aquaculture Pathology Laboratory, Dept. Veterinary Science and Microbiology, 1117 E. Lowell St., Tucson, AZ 85719, USA b. Corporación Centro de Investigación de la Acuacultura de Colombia, CENIACUA, Bogotá, Cra 9B-113-60 *Corresponding author

E-mail address: [email protected]; [email protected]

Abstract: Pacific white shrimp, Penaeus vannamei pre-exposed to Taura syndrome virus

(TSV) and then challenged with Yellow head virus (YHV) acquired partial protection from yellow head disease. Experimental infections were carried out using Specific-

Pathogen-Free (SPF) shrimp first exposed per os to TSV for different time frames: 27, 37 and 47 days (after feeding) and then challenged by injection with 1x104 copies YHV per shrimp (designated as TSV-YHV group). Shrimp not infected with TSV were injected with YHV as a control (designated as YHV group). Survival analyses between TSV-

YHV and YHV group were conducted and significant survival rates were found for all

50 the time frames (P<0.001). A higher final survival was found in the TSV-YHV group

(mean 55%) than in the positive control (0%) (P<0.05). Duplex RT-qPCR was used to quantify both TSV and YHV. Significant (5-6 times lower, P<0.001) YHV quantities

(3.52x109 copies µg RNA-1 from pleopods samples and 4.15 x109 copies µg RNA -1 from lymphoid organ samples) were found in TSV-YHV group in comparisons with the YHV group (1.88x1010 copies µgRNA -1 from the pleopods and 2.41x1010 copies µg RNA-1 from lymphoid organ samples). In situ hybridization (ISH) assays were conducted and differences in the distribution of the two viruses in the target tissues were found. Areas of

LO were infected with TSV but were not infected by YHV. This study suggests that a viral interference effect exists between TSV and YHV, which could explain, in part, the absence of YH disease in the Americas, where P. vannamei are often raised in farms where TSV is present.

Kew words: TSV, YHV, P. vannamei, Multiplex RT-qPCR, In situ hybridization

Introduction

Viral diseases are the most important cause of shrimp losses in commercial shrimp farming. Taura syndrome virus (TSV) and Yellow head virus (YHV) are considered two of the most pathogenic viruses present in shrimp farming in the Western and Eastern

Hemisphere, respectively. TSV (causative agent of Taura Syndrome disease, TSD) was first reported in Penaeus vannamei juveniles in Ecuador (Lightner et al. 1995). After it

51 emerged in Ecuador, TS spread through most Western Hemisphere shrimp culture regions and it later appeared in Southeast Asian countries in P. vannamei culture (Tu et al. 1999; Yu and Song 2000). At about the same time TSV was emerging in Ecuador,

YHV (causative agent of Yellow head disease, YHD) was reported in Asian shrimp farming countries. YHD was first reported in Thailand in 1990 in Penaeus monodon juveniles (Limsuwan 91; Boonyaratpalin et al. 1993), later on, YHD was reported in other countries, including India, Indonesia, Malaysia, the Philippines, Sri Lanka, Vietnam and Taiwan (Mohan et al. 1998; Wang & Chang 2000). YHV has been detected in the

Western Hemisphere, determined by RT-PCR. Nunan et al. (1998b) and Durand et al.

(2000) found YHV in imported frozen commodity shrimp from Asia. In 1999 some samples from an Ecuadorian farm, showed a positive RT-PCR result for YHV (Cenaim

2000). Recently YHV was reported in the Pacific coast of Mexico in shrimp from several farms (De la Rosa-Velez et al. 2006).

TSV is a non-enveloped, icosahedral virus that contains a single-strand positive sense

RNA genome of 10,2kb, and it belongs to the Dicistroviridae family (Bonami et al. 1997;

Mayo 2005). TSV contains two open reading frames (ORFs). ORF1 encodes the non- structural proteins: a helicase, a protease, and a RNA-dependent-RNA polymerase

(RdRp). The ORF2 encodes three capsid proteins CP1 (40kDa), CP2 (55 kDa), and CP3

(24 kDa) (Mari et al. 2002). YHV is an enveloped, positive sense, ssRNA genome virus of 26.6 kb that belongs to the genus Okavirus within the Roniviridae family (Cowley and

Walker 2002; Walker et al. 2005). YHV contains four ORF. ORF 1 encodes for the

52 protease, helicase, and RdRp. ORF 2 encodes for nucleocapside. ORF 3 encodes for the glycoprotein (gp 116 and gp64) and it is not known what ORF 4 encodes (Sittidilokratna et al. 2008).

One of the common characteristics of YHV and TSV is that they share many of the same target tissues. During acute phase, TSV and YHV infect cuticular epithelium (CE) of foregut and hindgut, stomach, gills and pleopods. Unlike TSV, YHV during acute phase targets also other organs such as the lymphoid organ (LO), connective tissue and hemocytes (Boonyaratpalin et al. 1993; Lightner 1996). During the chronic phase of infection, TSV and YHV produce LO spheroids in infected shrimp (Hasson et al. 1999a;

Anantasomboon et al. 2008).

Being that TSV and YHV viruses share similar target tissues and because TSV was already present in the Americas at about the same time YHV was becoming pandemic in

Asia, it is possible that TSV prevented shrimp from becoming infected with YHV during one of the several times in which frozen material from Asia infected with YHV and

WSSV (Nunan et al. 1998b) only resulted in the introduction of White Spot Disease

(WSD) to shrimp farms in the Americas. Thus, the objective of this work was to evaluate the viral interaction in shrimp pre-infected with TSV and then challenged with YHV under experimental conditions.

Materials and Methods

Shrimp and viruses

The TSV isolate used in this study was obtained from Hawaii in 1994 (HW) and the

YHV isolate was obtained in 1993 from farm-raised Penaeus monodon in Thailand. Both viruses have been maintained by continuous transfer in a Specific-Pathogen-Free (SPF) line of P. vannamei, “the Kona line” (Pruder et al. 1995) and stored at -70oC at the

University of Arizona. The same line was used in this study. All the experimental infections were carried out at the UAZ Aquaculture Pathology Laboratory (APL) in the

University of Arizona.

YHV Inoculum

YHV infected tissue was prepared using frozen samples. Only frozen YHV-infected shrimp P. vannamei heads, excluding the hepatopancreas, were homogenized in a buffer

(0.02 M Tris-HCl, pH 7.4, 0.4 M NaCl buffer; 1g/10 ml), using a tissue blender and clarified at 3500 x g for 20 min and 5000 x g for 20 min at 4oC. Samples were diluted

1:20 in 2% saline, aliquoted and frozen.

YHV lethal dosage

A titration with YHV was carried out to determine the highest dose in which mortality between 50 and 100% occurred. Eight 60 L aquaria with 10 SPF juvenile shrimp were set up in this study. Four different YHV inocula were tested in duplicate (1x102 through

1x105 viral copies per shrimp, at a 10 fold series dilution) by a single injection (100μl). In

Fig. 1 the final survival is shown 14 days post infection.

Pre-infection with TSV

Two hundred SPF P. vannamei, were stocked in a 1000 L round tanks and challenged with TSV per os by feeding the minced infected tissue at 10% of total body weight for one feeding.

Injection of YHV to TSV-pre-infected shrimp

For the experimental infection of shrimp pre-infected with TSV and then challenged with YHV (TSV-YHV group), three groups in duplicate were set up as a follow: 27 TSV days post infection (dpi), 37 TSV dpi and 47 TSV dpi, all with their respective negative control (shrimp injected with 2.5% saline) and positive control (shrimp infected only with

YHV; YHV group). In each 90 L tank, ten shrimp (mean weight: 5 g) were placed. The salinity and the temperature were adjusted at 25 ppt and 26±1oC respectively. Shrimp were infected with YHV inoculum by a single injection of a 1.0x104 copies of YHV per shrimp in a 100µL volume. Mortality was recorded twice per day from the start of the experiment. Cephalothoraxes of moribund shrimp were fixed in Davidson’s AFA fixative

(Bell and Lightner 1988) for H&E histological analysis. Moribund and recent dead animals were immediately frozen and stored at -70°C.

RNA extraction

Pleopods from the first abdominal segment were removed from each shrimp to determine the TSV and YHV viral load. Some samples of the lymphoid organ (LO) and adjacent areas were taken to determine the viral load as well. Total RNA was extracted using the RNeasy extraction kit (Qiagen) according to the manufacturer’s recommendations. The concentration of extracted RNA was determined by measuring the optical density (OD) at 260 nm.

Duplex TSV and YHV RT-qPCR

The real-time RT-PCR primers/TaqMan probe for TSV RT-qPCR (TSV1004F: 5-TTG

GGC ACC AAA CGA CAT T-3’, TSV1075R: 5’-GGG AGC TTA AAC TGG ACA

CAC TGT-3’, TSV-P1: FAM-5’-CAGCACTGACGCACAATATTCGAGCATC -

BHQ1) have been described previously (Tang et al. 2004). The primers and TaqMan probe for YHV (YHV141F: 5'-CGT CCC GGC AAT TGT GAT C-3’, YHV206R: 5'-

CCA GTG ACG TTC GAT GCA ATA-3’, YHV-P1: MAX NHS ester-5’-CCA TCA

AAG CTC TCA ACG CCG TCA -BHQ1) were designed from the conserved region,

ORF1, of the viral genome. The duplex RT-qPCR was carried out in a 25 µl reaction mixture containing 12.5 µl of TaqMan one-step RT-PCR master mixture (Applied

Biosystems), 20 ng of extracted RNA, each primer at a concentration of 0.3 µM, and the

TaqMan probe at a concentration of 0.1 µM. The RT-qPCR was performed on the

Mastercycler ep realplex (Eppendorf) with the following conditions: 48oC, 30 min for reverse transcription and 10 min at 95oC, followed by 40 cycles of 95oC for 15 s, and

60oC for 1 min. Amplification data were collected and analyzed with the Realplex 2.0 software (Eppendorf). The detection and quantification of TSV and YHV were from channels detecting FAM and JOE, respectively.

In situ Hybridization (ISH)

For ISH, histological sections of 4-5 μm thickness were prepared from Davidson’s fixed and paraffin-embedded shrimp tissues. Digoxigenin-labeled probes and colorimetric detection of the anti-DIG Fab fragment (alkaline phosphatase conjugate) with NBT and

X-phosphate was used. For YHV and TSV ISH assays, the protocol developed by Tang and Lightner (1999) for YHV and Mari et al. (1998) for TSV were used.

Statistical analysis

Statistical analyses were conducted in STATA IC10. The survival analysis and the cumulative survival probabilities were determined by Kaplan-Meier survival analysis.

Bartlett test was used to determine the equal variances. One-way ANOVA (α=0.05) was used to determine if statistical differences existed in viral load among groups and in the final survivals.

Results

Generation of TSV chronically infected shrimp

Penaeus vannamei were infected with TSV through the laboratory challenge, two weeks after exposure to TSV, 28% of the infected shrimp survived and went through a typical transition phase characterized by the multifocal melanized areas in the cuticle of the cephalothorax and abdomen. After molting, no further clinical signs were observed as the

TSV challenged shrimp became chronically infected.

YHV Titration

To determine the YHV titer for the challenge study, four inoculua were tested. The results showed that the injection of 1 x 102, 1 x 103 , 1 x 104 , and 1 x 105 copies of YHV per shrimp resulted in 0 %, 60%, 95% and 100% mortalities, respectively. (Fig. 1). Thus, inoculum containing 1 x 104 copies of YHV per shrimp was used in the challenge tests to evaluate if TSV-chronically infected shrimp were protected from infection by YHV.

YHV Challenge test in the TSV-infected shrimp

The challenge test showed differences in the final survival and survival curves among the groups exposed previously with TSV and then challenged with YHV (TSV-YHV) in comparison with the positive control infected only with YHV (Fig. 2a, b, & c).

The negative control showed a final survival of 100 %. The positive control (YHV only) showed a final survival of 0% in all three groups. Significant differences in survival were found among the three groups that had been pre-exposed to TSV for 27, 37 and 47 dpi prior to challenge with YHV (P<0.05). Analysis of the survival values using Kaplan-

Meier show significant differences in the survival curves (P<0.001).

Quantification of viral load by RT-qPCR

The YHV quantities between TSV-YHV (chronically infected TSV injected with YHV) and YHV (positive controls) groups were determined and compared by a duplex RT- qPCR for TSV and YHV. A 5 times higher YHV viral load was found in pleopods in the positive control group than in the TSV-YHV group (1.88x1010 versus 3.52x109 YHV copies μgRNA-1 )(P<0.001) (Fig. 3). The YHV copy number in LO of the TSV-YHV was 5.8 times lower than in the positive control (4.15x109 copies μgRNA-1 in the TSV-

YHV group versus 2.41x1010 copies μgRNA-1 in the positive control), values that were significantly different (P=0.01) (Fig. 3).

On the other hand, the TSV viral load in pleopod samples showed similar results after 27,

37 and 47 TSV d.p.i (p>0.05) with a mean of 9.25x107 copies of TSV μgRNA-1. The mean TSV copy number in LO was 5.78x108, and this was significantly higher that that in pleopods (P<0.005). This TSV distribution has been reported previously (Tang et al.

2004), and it is related to the typical TSV chronic phase where most of the TSV viral particles are harbored in this organ and circulating in the hemolymph (Poulos et al. 2008).

When comparing the TSV and YHV viral loads of dead versus surviving shrimp from the same group, a higher copy number of YHV was found only in the dead shrimp (3.52x109 versus 8.91x108 µgRNA-1)(P<0.05) suggesting that the mortalities observed were caused by YHV and not by TSV.

The results from the ISH confirmed that the shrimp were infected with the two viruses.

However, as expected, the distribution of the viruses was different. TSV presence was restricted to the LO whereas YHV was found in the cuticular epithelium (CE), connective tissue and LO (Fig 4). Interestingly, TSV distribution in the LO seems to be present mainly in the LO tubules, and YHV seems to be present in areas where TSV was not present and adjacent areas of the tubules including LO spheroids (LOS). The typical severe necrosis caused by acute YHV is not observed in the LO in shrimp pre-infected with TSV (Fig. 4). In Fig. 4G, 4H and 4I, TSV is not present in CE, and only YHV is present. Which is consistent with the TSV chronic phase and YHV acute phase.

Discussion

In this study a clear effect of viral interference was observed when shrimp were pre- exposed to TSV and then infected with YHV. This interaction of TSV on YHV infection reduced the mortality and, hence, an increase in survival was observed. The higher final survival in the TSV-YHV group is explained in part by the lower YHV viral load in pleopods and LO in the TSV-YHV group. In addition, the restriction of YHV distribution

60 in areas of LO where TSV was not present may have prevented this organ from the severe necrosis that was observed only in the YHV positive control group.

Previous studies have shown a correlation between viral copy number and higher mortalities. Srisuvan et al. (2006) found in TSV studies that the higher mortalities of

Belize-TSV strain were associated with the higher viral load present in the TSV-infected shrimp during TSV challenge test. In our study, the YHV viral load in LO and pleopods of the positive control was significantly higher that than in the TSV-YHV group, thus this could explain, in part, the higher mortality in the positive control infected only with

YHV.

It is known that YHV and TSV have some common target tissues such as cuticular epithelium in acute phase infections. TSV in the acute phase does not infect the LO. Only during the transition and the chronic phases, is a strong positive reaction observed by ISH

(Hasson et al. 1999b; Poulos et al. 2008). At that time, a higher viral load is present in comparison with pleopods (Tang et al. 2004). Unlike TSV, target tissues for YHV during the acute phase are the LO, hematopoietic tissue, connective tissue and hemocytes

(Lightner 1996). YHV copy number was lower in pleopods and LO in the TSV-YHV group indicating that YHV was not able to infect effectively tissues pre-infected with

TSV such as LO and CE. By histological analysis, similar results were observed. In the

TSV-YHV group, YHV was present, however, the typical severe necrosis of the LO was not observed as it was observed in the positive control. The ISH results showed that the

61 infection by the two viruses occurred in the LO, which was also confirmed by the RT- qPCR analysis. The same tissue can harbor the two viruses, however, the regions of the

LO infected first by TSV were not or were only lightly infected by YHV. The interaction between TSV and YHV is probably due to TSV interfering with the YHV infection, which is supported by the lower copy number of YHV in the TSV-YHV group, which may prevent the shrimp from severe acute necrosis of the LO, which in turns reduced the mortality.

Viral interaction has been reported for other shrimp viruses such as WSSV and IHHNV.

These two viruses share the same or similar target tissues as well (CE, connective tissue and hemocytes). Shrimp pre-infected with IHHNV and then challenged with WSSV are partially protected from WSD probably because IHHNV blocks the entry of WSSV, possibly by down-regulation of the viral receptor(s) (Tang et al. 2003) or by competition for common receptors (Bonnichon et al. 2006). In insects, similar phenomena have been reported in some cell lines. C6/36 Aedes albopictus cell lines infected with AalDNV virus are protected from Dengue disease (Burivong et al. 2004).

In some recent studies, it has been proposed that the cell receptor for TSV capsid protein

(CP2) is the laminin receptor (Lamr) (Senapin et al. 2006), and recently Lamr was found that it is also the cell receptor for YHV gp116 envelope protein (Busayarat et al. 2011).

Thus, this Lamr is the cell receptor for these two viruses. In our study, in shrimp pre- infected with TSV it is likely that either TSV occupies the cell receptors first, and thus

62 blocks the YHV infection in those areas. The competition between CP2 and gp 116 for the cell receptor, would limit the infection of the two viruses in the same region. In rainbow trout, it has been observed that the same cell receptor is used for two RNA viruses: Viral Hemorrhagic Septicemia (VHS) and Infectious Hematopoietic Necrosis

(IHN). In this study, VHS restrict the distribution of IHN to some tissues by competition for the cell receptors (Brudeseth et al. 2002). The difference in the viral load found in our study in the groups TSV-YHV versus the positive control (YHV group), support the hypothesis that the first pathogen excludes the infection by the second pathogen.

Some other possibilities may also explain the interference effect between TSV and YHV.

The resistance to YHV could be related to an unspecific mechanism involved in resistance against TSV that indirectly results in the resistance to YHV. However there is no evidence that this type of phenomena occurs in shrimp. There is one study in which interaction between TSV and IMNV occurs. Shrimp highly resistant to TSV are more susceptible to IMNV than the SPF control (White-Noble et al. 2010), which suggests that genes involved in the resistance of TSV are different those involved in IMNV resistance.

Some authors (Longyant et al. 2006; Chaivisuthangkura et al. 2008) reported that resistance to YHV was found in shrimp that are able to reduce expression of gp 116. It is possible that survivors from TSV infection have the ability to suppress expression of attachment protein such as CP2 in the case of TSV and gp 116 in the case of YHV.

YHV has been suspected to be present in some locations in the Western Hemisphere where shrimp are cultured, and sporadic positive results by RT-PCR suggest the presence of this etiological agent. Nunan et al. (1998b); Durand et al. (2000) found YHV in samples from imported commodity shrimp from Asia. In 2006, YHV was reported in the

Pacific coast of Mexico in wild shrimp (Castro-Longoria et al. 2008) and in raised shrimp (De la Rosa-Velez et al. 2006; Sánchez –Barajas et al. 2009;) and it was proved that this strain was infectious (Cedano-Thomas et al. 2010). Thus, one of the possible explanations about the absence of YHD in P. vannamei culture in the Americas could be attributed to the presence of TSV. TSV is currently endemic in many Latin-American shrimp farming areas (Lightner 1996; Lightner 2011), thus shrimp pre-exposure to TSV might block the entry of YHV by competing for the cell receptor. Even though YHV can still infect other tissues which TSV cannot, it is likely that the critical tissues, such as LO and CE are essential for YHV pathogenesis. Thus, by blocking the entry into those tissues, prevention of virus infection of other target tissues could be achieved. Additional work is needed to elucidate the whole blocking process at molecular level.

Acknowledgements

This work was supported by the Gulf Coast Research Laboratory Consortium Marine

Shrimp Farming Program, USDA Grant No. USMSFD 2010-38805-21115, the NOAA,

Saltonstall-Kennedy Grant No. NA09NMF4270102, and a special grant from the

National Fisheries Institute. We thank to Brenda Noble, for providing technical assistance and Rita Redman for histological services.

Tables and Figures

Fig. 3.1 YHV titration study in Penaues vannamei challenged by IM injection with 4 different concentrations. Each YHV concentration was done in duplicate. SE ± is shown.

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Fig. 3.2 Cumulative survival of SPF Penaeus. vannamei pre-exposed to TSV that were challenged with YHV (1x104copies/shrimp). TSV-YHV group (solid line), YHV positive control (dashed line) and negative control (dotted line) are represented. a)

27 TSV dpi, b) 37 TSV dpi. C) 47 TSV dpi. Data shown are the average of two replicates. Error bars represent the 95% confidence interval of the data set.

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Fig. 3.3 Comparison of YHV copy number in pleopods and LO of dead shrimp exposed only to YHV vs. shrimp pre-exposed to TSV and then challenged to YHV.

Error bars represent the 95% confidence interval of the data set.

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Fig 3.4 Histology of SPF Penaeus vannamei from infectivity studies with TSV and then challenged with YHV. A, D and G show the results of TSV ISH. B, E and H show the results of YHV ISH. C, F and I show H&E stained sections. A through F shows LO sections and G through I shows stomach cuticular epithelium section. Scale bar=50

μm

CHAPTER 4

USE OF A MARINE BACTERIUM (Vibrio sp.) AS A DELIVERY VEHICLE FOR

WSSV VP28 GENE INTO SHRIMP Penaeus vannamei

Fernando Arangurena,b*, Kathy FJ Tanga, Linda M Nunana and Donald V Lightnera

a. The University of Arizona, Aquaculture Pathology Laboratory, Dept. Veterinary

Science and Microbiology, 1117 E. Lowell St., Tucson, AZ 85721, USA

b. Corporacion Centro de Investigacion de la Acuacultura de Colombia, CENIACUA,

Bogota, Cra 9B-113-60, COLOMBIA

*Corresponding author

E-mail address: [email protected], [email protected]

Abstract: The White spot syndrome virus (WSSV) VP28 gene was inserted into a plasmid (pGFPuv) and transformed into a Vibrio sp., a marine bacterium. The transformed bacteria containing pGFP-VP28 was mixed with shrimp feed and was orally delivered to shrimp (Penaeus vannamei). During a 50-day feeding experiment, the VP28

DNA was detected in the hepatopancreas (HP) of infected shrimp. A 100% survival and

70 absence of histological lesions suggests that these bacteria are not pathogenic to P. vannamei. In a colonization study, this Vibrio sp. demonstrated its ability to colonize the

HP of the experimental shrimp.

The production of the recombinant VP28 protein by Vibrio sp. was confirmed by 12%

SDS-PAGE and Western blot assays. A challenge with a low dose of WSSV that was near its LD50 was conducted with the prototype “vaccine.” The genetically modified

Vibrio sp. with the pGFP-VP28 insert was mixed with feed pellets for oral administration. After exposure to WSSV, the final survival of the non-vaccinated control was 48.5%, while final survival in the “vaccinated” shrimp was 100%. These promising results indicate that the genetically modified Vibrio sp. has potential for use as a delivery vehicle for WSSV “vaccination” in cultured shrimp.

Keywords

WSSV, vaccine, VP28, Penaeus vannamei

Introduction

Among the shrimp diseases, White spot syndrome virus (WSSV) is the most pathogenic virus that has appeared in the shrimp industry worldwide. This virus was first reported in

China in 1992 and from there it spread to all of the Asian countries that cultured Penaeus

71 spp. (Takahashi et al. 1994; Chou et al. 1995). In 1995, WSSV was reported in the USA in imported frozen shrimp from Asia (Nunan et al. 1998b; Durand et al. 2000). By 1999-

2000, White Spot Disease (WSD) had become established in most of the major shrimp farming countries in the Americas and the disease caused significant losses to the shrimp farming industry (Lightner 2011).

WSSV is a large dsDNA enveloped virus that belongs to the genus Whispovirus in the family Nimaviridae (Vlak et al. 2005). The WSSV genome has been completely sequenced and is approximately 300 Kb, with 185 ORFs (Yang et al. 2001; Van Hulten et al. 2001a). To date, 50 structural proteins have been identified (Zhou et al. 2009), with four of them present in the viral envelope: VP19, VP24, VP26 and VP28 (Xie et al.

2006). Among these envelope proteins, VP28 is the most abundant (Van Hulten 2001b;

Rout et al. 2007), and is shown to play critical in the attachment and penetration of the virus into shrimp cells (Xie and Wang 2006; Yi et al 2004; Youtong et al. 2010).

Studies regarding the use of vaccination as a strategy to protect shrimp from WSD, have recently reported some promising results. The most common target proteins used to prevent shrimp from WSD are the envelope proteins VP28 and VP19. A short-term protection was afforded in P. vannamei when VP28 DNA was injected intramuscularly and then the test shrimp challenged with WSSV (Rout et al. 2007; Rajesh et al. 2008, Li et al. 2009). Similar results have been found using the purified VP28 protein through intramuscular injection into the shrimp that were then challenged with WSSV (Rout et al.

2007; Wei and Xu 2009). This vaccination process appears to prevent the infection by blocking the virus from attachment to the shrimp cell receptors instead of activating the shrimp immune response (Witteveldt et al. 2004; Rout et al. 2007). Although some of these experiments have shown protection, injection of the DNA or protein is not a feasible method in pond culture.

Recently, the use of attenuated bacteria have been tested as a potential delivery system for vaccination. Bacteria such as Escherichia coli (Witteveldt et al. 2004; Wei and Xu

2009), Bacillus subtilis (Fu et al. 2008) and Salmonella typhimurium (Ning et al. 2009) have been use as delivery vehicles that carry the vp28 gene and introduce it into the shrimp to protect them from WSD. The limitation of using these bacteria is the fact that they are all fresh water bacteria that cannot grow as normal micro flora in the shrimp digestive tract.

Vibrios are Gram-negative bacteria, present in high concentrations in estuarine and marine environments worldwide (Thompson et al. 2004). Vibrios are part of the normal bacterial community associated with the shrimp hepatopancreas (HP) and gut, in some cases composing up to 85% of total bacteria present (Moss et al. 2000). Even though a few species has been reported as shrimp pathogens, most species act as opportunistic bacteria (Lightner 1996). Various studies have shown that some vibrio species are able to be used as a probiotic bacteria, that when present in high concentrations compete and

73 colonize more efficiently than the pathogenic bacteria in the shrimp digestive tract

(Gullian et al. 2004; Gomez-Gil et al. 2002; Vandenberghe et al. 1999).

In this study we used a non-pathogenic marine Vibrio sp. that are adapted to the same environmental conditions of salinity and temperature as cultured shrimp. These bacteria were used as a vehicle to deliver the WSSV vp28 gene into the shrimp digestive system and produce the recombinant protein rVP28 and protect the shrimp from WSD.

Materials and Methods

Shrimp

Specific-Pathogen-Free (SPF) Penaeus vannamei, “the Kona line” (Pruder et al. 1995) was used in this study. All of the experimental infections were carried out at the

Aquaculture Pathology Laboratory at the University of Arizona (APL-UA).

White spot syndrome virus

The WSSV isolate used in this study was from a WSSV epizootic in China during 1993.

This virus has been maintained by continuous transfer in SPF P. vannamei and stored at -

70oC at the APL-UA.

Viral titration and qPCR

WSSV-infected shrimp P. vannamei heads, excluding the hepatopancreas, were homogenized in a buffer (0.02 M Tris-HCl, pH 7.4, 0.4 M NaCl buffer; 1g/10 ml), using a tissue blender and clarified at 3500 x g for 20 min and 5000 x g for 20 min at 4oC. The tissue homogenate were diluted 1:20 with 2% saline, aliquoted and frozen at -70oC.

Approximately 25-30 µl of inoculum was used for DNA extraction by using the High pure PCR template preparation kit (Roche Diagnostics) following the manufacturer’s protocol. Quantitative PCR was conducted using the protocol described by Durand and

Lightner (2002). 1x101, 1x102, and 1x103 WSSV copy number were tested to determine the lowest dose in which high mortality occurred (90-100%).

Vibrio sp.

From the Vibrio collection at APL-UA, the isolate, Vibrio 434 used, was originally isolated from the digestive tract in shrimp samples from Ecuador. This isolate was selected as a candidate vehicle to deliver the rVP28. Identification of the bacterium by two methods was conducted: by the biochemical test API 20-NE, and by molecular sequencing using the 16S rRNA and the Gyrase B (gyrB) genes. In order to amplify the

16S rRNA, universal primers for bacteria were used: 16S rRNA F: (5’ CCG AAT TCG

TCG ACA ACA GAG TTT GAT CCT GGC TCA G 3’); 16S rRNA R: (5’ CCC GGG

ATC CAA GCT TAC GGC TAC CTT GTT ACG ACT T 3’) (Nunan et al. 2003) and the gyrB, primers are GyrB F: (5’ GAA GTC ATC ATG ACG TTC TG 3’), GyrB R: (5’

AGC AGG GTA CGG ATG TGC GAG 3’)(Yamamoto and Harayama 1995). PCR products were sequenced and analyzed with nblast at the NCBI website.

Vibrio colonization

To determine the colonization of Vibrio 434, the bacteria were grown in Tripticase Soy agar (TSA) supplemented with 2% NaCl (TSA+) for 24 h. The bacteria were scraped off the plate and suspended in Trypticase Soya Broth (TSB) supplemented with 2% NaCl

(TSB+) and mixed with the feed pellets (Rangen 35%, Buhl, Idaho). The final concentration was approximately 1x109 CFU/g shrimp feed. An aquarium with 20 juvenile shrimp was set up and the inoculated feed mixture was used for one feeding at a ratio of 10% wet biomass. At 12, 24 and 36 hours post-inoculation (hpi), 3 shrimp were removed to quantify the Vibrio 434 concentration. Each HP was excised aseptically and weighted and by serial dilutions, the CFU/g of each HP was determined in TSA+ after incubation for 24 h at 32oC. Based on the typical morphology and using biochemical tests, Vibrio 434 was differentiated from other bacteria present in the HP samples.

Construction and purification of plasmid

DNA extracted from hemolymph of a WSSV-infected shrimp was used as a template to amplify the complete ORF of vp28 with specific primers: VP28 F (5’ GCG AAG CTT

AAT GGA TCT TTC TTT C 3’) including a Hind III restriction site (underlined) and

VP28R: (5’ GAC ACA TCT AGA TAC TCG GTC TCA G 3’) containing a Xba I restriction site (underlined). The insert was cloned in a pGEM-T vector (Promega) and

76 then the VP28 DNA fragment was excised and cloned in pGFPuv vector (Clontech) by digestion with Hind III and Xba I. The resulting recombinant plasmid pGFPuv-VP28 and the control pGFPuv were transformed in E. coli JM 109 competent cells for propagation.

The plasmids were purified using the plasmid purification kit (Omega) according to the manufacturer’s instructions. The recombinant plasmid pGFPuv-VP28 was verified by

DNA sequencing using an automated DNA sequencer, ABI Prism 3730xl (Applied

Biosystems). The concentration of pGFPuv-VP28 and pGFPuv were determined by measuring the optical density at 260 nm.

Transformation of plasmid pGFPuv-VP28 into Vibrio 434.

The transformation of Vibrio 434 was conducted following the protocol described by

Hamashima et al. (1995) and Wang and Griffiths (2009). In brief, Vibrio 434 was grown

+ in (TSA ) until early log phase (O.D600 nm = 0.4). In order to make the cells competent, three-washes with EP buffer (272 mM sucrose, pH 7.4) and three centrifugations at 4000 x g at 4oC for 20 min were conducted. The purified plasmid pGFPuv-VP28 and pGFPuv were electro-transformed into Vibrio 434. using a 0.2 cm cuvette at 25 µF, 1.5 kV and

200Ω (Gene pulser Bio-Rad II system). The positive transformants were selected in TSA+ plates supplemented with 100 µg/ml ampicillin (TSA+Amp+). Successful transformation was confirmed by the presence of glowing Vibrio 434 under UV-light and by PCR using specific primers for VP28.

In vitro stability of plasmid pGFPuv-VP28 in Vibrio 434.

The recombinant Vibrio was growth in TSA+ at 32ºC and propagated for about 550 generations (~5 days). In vitro plasmid stability was determined by two methods: the number of glowing CFUs, compared to the total CFUs in TSA+Amp- and by determining the ration between CFUs grown in TSA+Amp+ versus CFUs grown in TSA+Amp-.

Protein expression of the pGFPuv-VP28 plasmid into Vibrio 434.

From a TSA+Amp+ plate, one CFU was used to inoculate 5 ml TSB+Amp+, with shaking at 200 rpm until the OD at 600 nm of 0.6-0.7 was reached. Samples were divided into two tubes. In one tube, Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 µM. Both samples were incubated for 3 hours. 10µl and 100 µl were removed from each tube and centrifuged at 10000 rpm for 2 min. The pellets from each sample were suspended in 1X gel loading buffer, boiled for 3 min and centrifuged again. 30 µl of each sample was loaded in a 12%-SDS PAGE gel and run for 45 min at

150 V. The proteins present in the gel were transferred onto a nitrocellulose membrane at

1mA/cm2. Western blot analysis was conducted according to Poulos et al. (2001). An anti-VP28 monoclonal antibody (Aquatic, Stirling) at a concentration of 1:1000 was used.

Oral administration of Vibrio 434-pGFPuv-vp28 in shrimp P. vannamei and isolation of recombinant Vibrio 434

The transformed Vibrio 434-pGFPuv-vp28 was grown as described above. Two 60L aquaria with ten juveniles P. vannamei (~ 4 g) stocked in each, were fed with the coated feed once per day for 3 days at ratio of 10% wet biomass per aquarium. Salinity and temperature were adjusted to 25ppt and 26ºC, respectively. During the 10, 15, 40 and 50 d.p.i, shrimp were removed and samples of HP was taken and preserved in 95% ethanol.

The primers for VP28 DNA detection were used to detect the presence of the transformed bacteria by PCR. At the end of the challenge, on day 50 , shrimp were fixed in

Davidson’s fixative for histological analysis.

Oral vaccination with Vibrio sp-pGFPuv-VP28

P. vannamei juveniles (4 g) were tested in 6 tanks, 4 non-vaccinated control tanks and 2 vaccinated tanks, with 20 shrimp each. Shrimp were fed for 7 days with the coated feed containing Vibrio 434-pGFPuv-VP28 once per day at ratio of 10% wet biomass per tank.

On day 8, shrimp were injected with 10 copies of WSSV. After 21 dpi, the final survival was determined. Moribund shrimp were tested for WSSV by histology and PCR.

Statistical analysis

Statistical analyses were conducted in STATA IC10. Bartlett test was used to determine the equal variances. One-way ANOVA (α=0.05) was used to determine the difference of final survival among groups.

Results

WSSV titration and qPCR

The lowest dose, at which 100% mortality occurred, was 10 copies of WSSV. Thus, this viral load was used for the experimental infections.

Vibrio 434 Identification.

This Gram-negative rod, was grouped among the Vibrio species according to the biochemical test results. The API-20NE number, 7147744, was considered by the system as a low discrimination identification. The 16SrRNA gene sequence from Vibrio 434 that was blasted in NCBI database gave similar results (GenBank accession number:

JN628995). The results are shown in the Table 1.

All of the bacteria that had the highest identity with Vibrio 434 were also Vibrio spp.

Analysis of the GyrB gene from Vibrio 434 revealed similar results (GenBank accession number: JN628994). The highest identity was found with V. xuii, with a maximum identity of 100% followed by Vibrio sp. with an 86% identity. Hence, it is likely that

Vibrio 434 is new species of Vibrio.

Vibrio colonization

The results from the colonization studies are shown in Table 2. Three types of bacteria grew in TSA+ with the most abundant species being Vibrio 434. The proportion of Vibrio

434 compared to the total bacteria count decreased from 88.2% at 12 hpi, to 42.1% to

36.hpi.

In vitro stability of plasmid pGFPuv-VP28 in Vibrio sp.

The doubling time of Vibrio 434 was calculated to be about 13 min. Thus, it was propagated for about 554 generations (5 days) in TSA+Amp- and TSA+Amp+ at 32oC (Fig.

1) .

After approximately 200 generations, the percentage of bacteria containing the plasmid was high (~70%), then the percentage of transformed Vibrio decreased to 11% by the 554 generation. Even though the percentage is relatively low, the CFUs at day 5 was about

6.8x108CFU/ml of broth.

Oral administration of shrimp with Vibrio sp-pGFPuv-vp28

At the end of the WSSV challenge of P. vannamei fed with Vibrio 434-pGFPuv-vp28, the final survival was 100%. The hepatopancreas samples taken during the course of the infection were analyzed by PCR (Fig. 2).

The HP samples analyzed after the inoculation with Vibrio 434 up to the 50 dpi show the presence of the vp28 amplicon (615bp). This indicates that the recombinant plasmid were present in the HP of the shrimp.

Protein expression of recombinant plasmid in Vibrio 434

The results of the 12% SDS-PAGE (left) and the Western blot (right) are shown in Fig. 3.

The 57 kDa band in the Western blot that reacts with the VP28 Mab, corresponds to the rVP28-GFP fusion protein present in the sample. This result confirmed that the band corresponded to the recombinant fusion protein.

WSSV Challenge bioassay

The challenge test results revealed the protection afforded by the recombinant fusion protein rVP28-GFP, based on final survival; 100% of the vaccinated population, contrasted to 48.5% in the non-vaccinated group (Fig. 4).

Shrimp from the vaccinated and non-vaccinated groups were analyzed by histology. The typical WSSV lesions were observed in the non-vaccinated group, but not in the vaccinated population.

Discussion

In this study, we demonstrated, for the first time, the ability of the marine bacterium

Vibrio 434 to be used as a vehicle to orally deliver a shrimp recombinant vaccine against

WSSV. In previous studies, intramuscular injection of a specific DNA vaccine had been used to partially protect WSSV-infected shrimp from WSD. However, this method is not feasible in shrimp culture at a large scale. Because the delivery route may be the most

82 important aspect of vaccination in shrimp, in this study we decided to use a marine bacterium isolated from a healthy shrimp to incorporate the WSSV vp28 gene to produce the rVP28. Adding the bacteria containing the recombinant protein to shrimp feed pellets protected the shrimp from WSD in a subsequent challenge.

The safety of Vibrio 434 was demonstrated after exposing shrimp for 3 days to high bacterial doses, and then analyzing the shrimp at 50 dpi. In these shrimp an absence of histological lesions and 100% final survival was obtained. This result was not surprising because Vibrio spp. are the most abundant heterotrophic bacteria present in the shrimp habitat and they form part of the natural micro flora in the gut and hepatopancreas

(Yasuda and Kitao 1980; Hisbi et al. 2000; Gomez-Gil et al. 2002). Moss et al. (2000) reported that Vibrios spp. can form up to 85% of total bacteria present in shrimp gut. In our study, the colonization of Vibrio 434 in the shrimp HP was assessed. After 36 hpi, the

CFU were 2.75x105/g of HP, which is a relatively high value. Gullian et al. (2004), using a probiotic Vibrio P63, found a colonization after 24 hours post- infection (hpi) of

3.4x104 CFU/g HP, which was lower than our results. Even though the proportion of

Vibrio 434 in comparison to the total bacterial counts was lower at 36 hpi (Table 2), the transformed bacteria were still the dominant bacteria in the HP of the inoculated animals.

Interestingly, even though there were high CFU in HP, neither clinical signs nor histological lesions were found that could be considered to be due to Vibrio 434. This indicates clearly that this is a non-pathogenic bacterium that is able to colonize efficiently the shrimp HP.

The presence of VP28 DNA was evidenced in the HP samples after 50 dpi. This indicated that the bacteria carrying the plasmid were present in the HP. The recombinant VP28 protein production was confirmed in vitro, indicating that the bacteria were able to make the recombinant protein (Fig 4).

One of the advantages about Vibrio 434 is its ability for rapid growth in simple media such as TSA+. The doubling time was calculated to be about 13 min, which is higher that

E.coli, B. subtilis and S. typhimurium, and thus Vibrio 434 can colonize the HP of a shrimp faster. At the same time, the expression of the rVP28 into the HP would be favored.

For the challenge an intramuscular injection was used to ensure that each shrimp received the same dose. In this study the vaccinated shrimp showed better protection when compared to the non-vaccinated group. However, because of the low dose used, some shrimp from the non-vaccinated group may not have received an infectious dose of

WSSV and did not become infected. This variability in the final survival did not allow us to see significant differences.

The mechanism by which the rVP28 protect shrimp from WSD is not clear. Some studies reported that VP28 stimulates the shrimp innate immune system to produce antimicrobial peptides or increase the levels of some immunological parameters such as lysozyme, alkaline phosphatase, phenoloxidase and total superoxide dismutase (Li et al. 2010; Wei

84 et al. 2009; Kumar et al. 2008). However, another hypothesis is that the rVP28 blocks the

WSSV from entry to the shrimp cells (Witteveldt et al. 2004; Rout et al. 2007). This hypothesis is supported by some recent studies in which it was found that the small

GTPase protein (Pmrab-7) is the cell receptor for the WSSV-VP28 (Sritunyalucksana et al. 2006). Thus, if there is a high presence of rVP28 in the shrimp environment, some of these proteins would bind to the Pmrab-7 receptors and by occupying them, the WSSV virions would not be able to attach to the receptors bound to the rVP28 proteins and therefore, the infection would not take place.

Because Vibrio spp. are normal inhabitants of marine and estuarine systems where shrimp are raised, it is likely that the method reported here could be used in areas where

WSD is endemic by mixing bacteria with shrimp feed pellets at a commercial scale and thus prevent or delay WSD. However, further studies are needed to quantify the amount of rVP28 proteins required to control WSD.

Acknowledgements

This work was supported by the Gulf Coast Research Laboratory Consortium Marine

Shrimp Farming Program, USDA Grant No. USMSFD 2010-38805-21115. We thank to

Brenda Noble, for providing technical assistance and Rita Redman for histological services.

Tables and Figures

Table 4.1 Comparison of the Vibrio 434 16SrRNA sequence identity (%) with top five sequences blasted in NCBI.

Bacteria GenBank No Origin Host Identity (%) Vibrio sp DQ146983.1 Australia Crab 99 V. gallicus AJ440009.1 Belgium Abalone 99 Vibrio xuii NR025478.1 China/Ecuador Shrimp 99 V. hepatarius EU834018.1 China Marine water 99 Vibrio sp. EF587960.1 Belgium No reported 99

Table 4.2 Vibrio 434 colonization in hepatopancreas (HP) samples. Each value is the mean of three replicates. Hour post CFU Vibrio 434/g inoculation CFU Total bacteria /g HP Vibrio 434 (%) HP (hpi) 12 1.51E+07 1.71E+07 88.2 24 1.34E+06 2.28E+06 58.7 36 2.76E+05 6.56E+05 42.1

Fig. 4.1 In vitro stability of the Vibrio 434 carrying the DNA plasmid pGFP-uv-VP28 after 554 generations.

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CHAPTER 5

QUANTIFICATION OF THE BACTERIAL AGENT OF NECROTIZING

HEPATOPANCREATITIS (NHP-B) BY REAL-TIME PCR AND COMPARISON

OF SURVIVAL AND NHP LOAD OF TWO SHRIMP POPULATIONS

Luis Fernando Aranguren*a,b, Kathy FJ Tanga, Donald V Lightnera

a. Department of Veterinary and Microbiology, University of Arizona, Tucson, 85721, USA b. Corporacion Centro de Investigacion de la Acuacultura de Colombia, CENIACUA, Carrera 9C No 114-60, Bogota, Colombia

* Corresponding author

Email: [email protected]; [email protected]

Abstract: A real-time quantitative PCR (qPCR) assay was developed using a TaqMan probe to detect and quantify the necrotizing hepatopancreatitis bacterium (NHP-B) in

Penaeus vannamei. A pair of primers which amplify a 67 bp DNA fragment and a

TaqMan probe were selected from the 16S rRNA gene of NHP-B genome. A positive control plasmid DNA was used to demonstrate that the NHP qPCR assay has a detection limit of 100 copies and a log-linear range up to 108 copies. An NHP-B challenge test

92 using two different populations, Colombia (COL) and Specific Pathogen Free KONA line

(KONA), was conducted. Higher final survival and a better survival curve were found in the COL population. No differences in NHP-B bacterial load were found in hepatopancreas (HP) analyzed from the survivors from each population. Sensitivity of the qPCR test was much higher than the conventional PCR (100 copies vs. 1x105 copies).

Key words

Necrotizing hepatopancreatitis, Penaeus vannamei, Real-time PCR.

Introduction

Necrotizing hepatopancreatitis (NHP) is a disease caused by an unclassified Gram negative, pleomorphic intracellular alpha proteobacteria which will be called NHP-B for the purpose of this paper (Frelier et al. 1992; Lightner and Redman 1994). NHP affects cultured penaeid shrimp in several countries from the western hemisphere including the

USA, most Central American and South American countries that farm shrimp (Lightner

1996), and it was recently reported in Eritrea (Lightner and Redman, submitted). NHP is a chronic disease that causes mortalities up to 95% in shrimp population in grow-out ponds (Lightner 1996; Johnson 1990) and broodstock ponds (Aranguren et al. 2006.

Morales et al. 2006). The occurrence of NHP is related to specific environmental conditions such as high temperature and high salinity (Lightner 1996; Vincent & Lotz,

2007). NHP-B-infected shrimp display a typical soft shell, flaccid bodies, lethargy,

93 reduced feed intake and empty midgut (Lightner 1996). Hepatopancreas (HP) lesions in acute phase include intense intracellular hemocytic response, a few to many melanized

HP tubules and necrosis and sloughing off of HP tubule epithelial cells. HP lesions in chronic phase include marked atrophy of tubules and reduced epithelial cell height, low lipid storage and intratubular edema (Lightner 1996).

Several detection methods have been developed to diagnose and confirm NHP-B presence including histology, in situ hybridization, PCR (Lightner 1996; Loy and Frelier

1996; Nunan et al. 2008) and immunohistochemistry (Bradley and Dunlop 2004). The only study in which NHP-B has been quantified, was developed by Vincent and Lotz

(2005), however, inconsistent results in bacterial load were found in our lab, hence the need to develop a new protocol.

In Colombian shrimp culture, NHP disease is rarely reported from commercial ponds, even when the temperature and salinity are in the range generally considered favorable for disease development. Furthermore, in recent years in Colombia, the signs of NHP-B infection have been restricted to ponds or tanks in which broodstock are raised and matured (Briñez et al. 2003, Aranguren et al. 2006). It is noteworthy that Colombian selection at the level of multiplication of broodstock is carried out under conditions where diseases such as Vibrio spp. IHHNV and others that are common in commercial grow out, are not excluded.

The purpose of this study was to develop a qPCR protocol for the detection and quantification of NHP-B and evaluate the NHP-B resistance of two shrimp populations and to determine the NHP-B load in survivors. In addition, the sensitivity and specificity of qPCR and the conventional PCR assay were compared.

Materials and Methods

The NHP-B strain that used in this study, originally from Texas, was kindly provided by

Dr H. Harris (Iowa State University) and came from a long-term maintenance system

(Vincent et al. 2004; Crabtree et al. 2006). The NHP-B infected hepatopancreas (HP) was stored at -70°C in the Aquaculture Pathology Laboratory at the University of Arizona until used to start the infection. The species of shrimp used in this study was Penaeus vannamei (Boone). Shrimp Taxonomy used in this study was according to Holthuis,

(1980).

Quantitative PCR (qPCR)

Primer Express software version 2.0 (Applied Biosystems) was used to design primers and the TaqMan hydrolysis probe (Applied Biosystems) from the 16S rRNA gene of

NHP-B (GenBank U65509) (Loy et al. 1996). Primers NHP1300F: 5’-CGT TCA CGG

GCC TTG TAC AC-3’ and NHP1366R: 5’-GCT CAT CGC CTT AAA GAA AAG ATA

A-3’ were used to produce a fragment of 67 bp. The TaqMan probe NHP: 5’-CCG CCC

GTC AAG CCA TGG AA-3’, which corresponds to the region from nucleotides 1321-

1340, was synthesized and labeled with fluorescent dyes 6-carboxyfluorescein (FAM) on the 5’ and N,N,N,N-tetramethyl-6-carboxyrhodamine (TAMRA) on the 3’ end.

Based on optimization of primers and TaqMan hydrolysis probe concentration, the amplification reactions were performed in a final volume of 20 μl with 10 μl of PerfeCTa qPCR SuperMix (Quanta, Biosciences), 0.3 μM of each primer and 0.1 μM of TaqMan probe. The reaction mixture contained 0.4 μg of DNA. The qPCR profile consisted of 3 min at 95°C followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Amplification detection and data analysis for qPCR assays were performed with the master cycler

Realplex 2.0 (Eppendorf).

To generate a plasmid DNA standard for the NHP qPCR assay, the primers NHP F1237:

5’-TGC AAC TCG AGA GCA TGA AG-3’ and the NHP 1413R: 5’-CCC CAG TCA

TCA CCT TTT CT-3’ were used to generate an amplicon of 177 bp from the NHP 16S rRNA. The PCR product was cleaned using a QIAquick PCR purification kit (Qiagen).

The fragments were ligated to a pGEM-T Easy vector (Promega) and transformed into

Escherichia coli JM109 cells (Promega). The recombinant plasmid pNHP1 was verified by DNA sequencing with an automated DNA sequencer ABI Prism 3730xl (Applied

Biosystems). The concentration of pNHP1 was determined by measuring the optical density at 260 nm. Each sample was run in triplicate and average OD result was used to estimate the amount of DNA present in each sample. When analyzing the samples a pure water (Sigma, molecular biology reagent) blank was used between samples as a control.

Also, the 260:280 ratio was used to determine protein contamination. The DNA copy number was obtained by using the formula: (Amount of DNA in ng) (Avogadro’s number) / (650Da) (length of template as bp) (Staroscik, 2004). The sensitivity of the qPCR was determined using 10-fold dilutions of purified plasmid pNHP1. As carrier, a yeast tRNA was used in a concentration of 10 ng/μl (GeneReach Biotechnology). The concentration of plasmid DNA used ranged from 108 to 1 copy per reaction. The assay was repeated six times to demonstrate the reproducibility.

Experimental challenge

The challenge tests were carried out at the UAZ Aquaculture Pathology Laboratory

(APL) during 2008-2009. Used in these tests were Taura Syndrome Virus (TSV)-Specific

Pathogen Resistant (TSV-SPR) P. vannamei from Colombia and designated as the COL line. Also used was a Specific-Pathogen-Free (SPF) line of P. vannamei, designated as the KONA line (Pruder et al. 1995), and obtained from the Oceanic Institute. The average weight of the shrimp from both lines at the start of the test was 2.8g±0.2g. Shrimp were tagged with fluorescent elastomer tags (Northwest Marine technologyTM) in 6th abdominal segment. Fifty shrimp from each population were stocked into each of three 1000 L tanks. Two were treatment tanks while one tank was used as a control. Temperature was adjusted to 30oC and salinity to 30 ppt, which are considered to be optimal conditions for the development of NHP-B disease (Lightner 1996, Vincent and Lotz 2007). Shrimp were infected with NHP-B inoculum generated as described before (Vincent et al. 2004;

Crabtree et al. 2006).

Shrimp were inoculated by reverse gavage. In brief, 10 infected shrimp were taken from the long–term maintenance system and their HP were excised and weighted. Then, HPs were combined with 2.5% saline in a ratio of 1:2, (w/v.) The mixture was ground until homogenized and then centrifuged at 6000 x g for 1 min. The supernatant was mixed with a commercial red food dye in a ratio of 1000:3 (v/v). Shrimp were starved for 24 hr prior to the infection process to avoid any obstruction by feces in the digestive tract. By using an automatic pipette, a 100 μL of inoculum was introduced in the shrimp anal cavity dispensing the inoculum slowly to avoid traumatic effects. The red dye present in the inoculum facilitated the observation of the introduction of the inoculum first into the hindgut, then into the midgut and hepatopancreas. The shrimp present in the control tank were inoculated by reverse gavage using a 0.85% saline solution. Starting 12 hr after the inoculation process, the shrimp were fed once a day with a commercial pelleted feed

(Rangen 35%, Buhl, Idaho).

Mortality was recorded daily from the start of the experiment. Moribund shrimp were fixed in Davidson’s AFA fixative (Bell and Lightner 1988) and analyzed by histology to verify their disease status. Dead animals were frozen and stored at -70°C for PCR analysis.

Seven times during the experiment, shrimp were removed from the experimental tanks in order to determine the survival at each time point for each population. Fecal samples

98 were taken weekly in each tank. At the end of the challenge (116 days post inoculation,

d.p.i), the HPs from all survivors were preserved in 95% (v/v) ethanol /dH2O molecular grade prepared from molecular grade 100% ethanol (Decon, Labs, Inc) for determination of NHP-B load in each population by qPCR analysis.

DNA extraction

DNA extraction was carried out for both populations from samples preserved in 95% ethanol. Approximately 25-50 mg of individual HP was used for DNA extraction by using the High pure PCR template preparation kit (Roche diagnostics) according to the manufacturer’s protocol and stored at -20°C until further analysis.

PCR

Oligonucleotide primers NHPF2: 5’-CGT TGG AGG TTC GTC CTT CAG T-3’ and

NHPR2: 5’-GCC ATG AGG ACC TGA CAT CAT C-3’, which generated a 379 bp amplicon, were used in the conventional PCR analysis (Nunan et al. 2008). The primer concentration (F2/R2) used for each was 0.31μM. PCR was conducted in a final volume of 25 μL. PuRetaq ready- to-Go PCR beads (GE Healthcare) was used. The cycling parameters were as follows: 1 cycle at 95°C followed by 25 cycles at 60°C for 30 s, 72° at 30 s and 95°C for 30 s, and finishing with a final extension at 60°C for 1 min and 72°C for 2 min. All amplifications were performed in an Eppendorf Master cycler. Following amplifications, the PCR products were electrophoresed in 1.5% agarose gels containing

0.5 μg ml-1 ethidium bromide and visualized under ultraviolet light and digitally photographed by the AlphaImager (Alpha Innotech).

Statistical analysis

Statistical analyses were conducted in STATA IC10. Comparison of bacterial load mean±SE was determined by two sided t-test (α=0.05). The survival analysis was determined by Kaplan Meier survival analysis (Kaplan and Meier 1958).

Results

Analytical specificity of qPCR

The analytical specificity of the NHP qPCR was determined by using different microorganisms including NHP-B from two different origins (Texas and Mexico). DNA extracted from shrimp infected with viral diseases, including WSSV, IHHNV, HPV, and

MBV and bacterial diseases including Spiroplasma penaei, Vibrio spp., and a rickettsia- like bacterium isolated from Madagascar, were used as templates. Negative results were found with all of the other microorganisms. The only positive results were found in the

DNA samples that contained NHP-B from Texas and Mexico.

100

Analytical sensitivity of qPCR

To determine the analytical sensitivity of the NHP qPCR, a 177 bp NHP fragment containing the target sequence of qPCR was used as a standard by testing serial dilutions from 1x100 to 1x108 copies. It was found that at 10 copies of NHP-B were detected in two out of six assays, while 100 copies were detected in all six assays with a 0.5 mean

Standard Deviation (SD) (Table 1), thus the detection limit was considered to be 100 copies (Fig. 1). To determine the reproducibility of the NHP qPCR, six standard curves were compared over a 7-log range from 100 to 1x108copies per reaction (Table 1).

The SD within each run was between 0.0 to 1.1 in 102 copies and 0.1 to 0.3 in the 108 copies. For the combined data from the six independent runs, there was not a linear relationship between SD and the quantification cycle Cq values (before called threshold cycle) (Bustin et al. 2009), (p>0.05), showing consistencies in pippeting. For the inter- assay replicates, the SD values between 0.2 and 0.8 were found, indicating good reliability in this assay. The R2 of these six standard curves were always greater than

0.98, indicating good reproducibility, and also, the reaction efficiency values fluctuated between 0.97 through to 1.06 indicating good optimization of the protocol (Fig. 1).

Challenge test

NHP-B infection was confirmed in the infected tanks during the challenge test using histological sections of the hepatopancreas. Typical lesions of NHP-B infection, including hemocyte infiltration and granulomas in the HP with intratubular edema, were

101 observed in symptomatic shrimp. The animals sampled from the control tank tested negative to NHP-B by PCR and qPCR. The mortality peak was found to be between the

35 and 63 d.p.i. After 116 d.p.i, the shrimp from each population were counted and the final survival was determined (Table 2). The survival curve was similar in the two infected tanks (data not shown).

With similar survival patterns in the two infected tanks (p>0.05), the survival results data were pooled and analyzed by the Kaplan Meier survival analysis. The survival curve of the COL population was significantly greater than the KONA SPF population (p=0.0002)

(Table 2). The estimated time for 75% survival was 35 days in KONA line, compared to

77 days in the COL population. The final survival at the end of the study after 116 days was 41% in the KONA and 71% in COL population (Fig. 2).

Hepatopancreas and feces quantification

Fourteen shrimp per each population per each tank were analyzed by NHP qPCR. In tank

1, 100% of the survivors from the COL and KONA populations showed NHP-B in HP, whereas in tank 2, 78.5% of survivors from each population gave a positive qPCR assay for NHP-B. In tank 1, the NHP-B copy number in COL population ranged from 2.8x103 to 4.5x107 copies μg-1 of DNA were observed whereas in the KONA population the NHP-

B copy μg-1 of DNA ranged from 3.0x102 to 8.8x107. In tank 2 the NHP-B copy number presented in the COL population were from 0 to 3.1x102 copies μg-1 of DNA. In the

KONA population the range was from 0 to 3.3x102 copies μg-1 of DNA. There were no

102 significant differences in the mean NHP-B copy number between the populations in either tank (p>0.05) (Table 3). All fecal samples were positive for NHP qPCR in tank 1

(10/10) with a mean copy number of 4.2x106 copies μg-1 of DNA In tank 2, 83.3%

(10/12) of the samples were positive, with a mean copy number of 4.3x103 copies μg-1 of

DNA.

Comparison of NHP- qPCR vs. PCR

All the samples run by qPCR were also run by the conventional NHP PCR described by

Nunan et al. (2008). By qPCR, all HP samples were positive in tank 1, however by conventional PCR only 75% of these samples were positive. The mean copy number of negative samples by PCR was 2.6x103 copies μg-1 of DNA ranged from 3.0x102 through to 4.6x103, whereas the mean copy number in positive samples by PCR was 2.8x107 copies μg-1 of DNA and ranged from 5.3x103 through to 8.8x107. In tank 2, which showed higher survival, the percent of positive cases of NHP-B by qPCR in HP samples was

78.5%. By PCR only 14.2% of these samples were positive. The mean copy number was

1.2x102 copies μg-1 of DNA (Table 3).

Another approach to verify the difference in sensitivity between NHP qPCR and NHP-B

PCR, a positive sample with a known copy number (8.7x107 copies μg-1 of DNA) was the use of serial dilutions from 107 through to 101. This dilution series of samples was run by both PCR and qPCR. By qPCR all the samples were positive. The same samples run by

PCR showed that only those samples with a high bacterial copy number were positive

103

(8.7x107, 8.7x106 and 8.7x105) (Fig. 3A). Samples with 8.7x104 and lower were not detected with the NHP-B PCR assay (25 cycles).

After increasing the PCR cycles to 35, without changing any other parameter in the PCR protocol, an increase in the detection of NHP-B was observed (Fig.3B). Samples 3

(8.7x103) and 4 (8.7x104) that were negative in 25-cycle PCR, were positive after 35 cycles, indicating a 2 log increase in the sensitivity of the assay with this modification.

The same analysis was conducted with the HP samples from tank 1. After 35 cycles, five out of seven samples that were initially negative were found to be positive. The mean copy number of those five samples was 2.7x103.

Discussion

In this study, a quantitative PCR assay was developed to detect and quantify NHP-B in shrimp tissues. The detection limit of this qPCR is 1x102 copies. The sequencing from the pNHP1 shows 100% of identity with the fragment of the NHP 16S rRNA sequence reported by Loy et al. (1996). With NHP-B from two different geographical areas

(Mexico and Texas) and several other shrimp pathogens, the only positive results were found with the two NHP-B isolates, indicating the high specificity of this assay. The only study in which NHP-B has been quantified was developed by Vincent and Lotz (2005).

104

In the Vincent and Lotz (2005) study, a detection limit of 10 copies was reported when 5

μL of sample template DNA after 45 cycles was used. Moreover, when this assay was used in the present study, inconsistent results were found with copy number. In the

Vincent and Lotz protocol, samples that were strongly positive by conventional PCR

(detection limit about 105), gave a low copy number (between 0 and 5x102) with the qNHP-B protocol, suggesting underestimation of the bacterial copy number. Similar results have been found previously in our lab (B. Poulos, unpublished data).

Nevertheless, the reason for the low amplification efficiency with the Vincent and Lotz

(2005) assay is unclear.

No difference in final NHP-B load was found between COL and KONA in either of the two NHP-B challenged tanks (p>0.05). Very few studies have been focused on quantification of bacterial/viral load in shrimp populations. Srisuvan et al. (2006) found that after TSV challenge test in two populations, TSV resistant line and a susceptible SPF

KONA line, the TSV viral load was greater in the SPF KONA line than in the TSV resistant line. In contrast, in our experiment no difference in NHP-B load was found, possibly because the resistance mechanism in bacteria is different from viruses.

Even though that two treatment tanks were exposed to the same NHP inoculum, the bacterial loads in both HPs and feces were higher in shrimp from the tank 1(p<0.05). This was resulted from a more active bacterial multiplication and a heavy infection in the shrimp. The lower bacterial loads in tank 2 may be related to a longer prepatent phase

105 and thus the mortality was delayed. Tagging two populations differently in the same tank allows for a direct comparison between populations regardless different infection levels between tanks.

In a 60 day-NHP-B challenge test using a SPF Kona line, survival was between 0-30% after infection with either fresh or frozen preparations of NHP-B (Crabtree et al. 2006).

This compares with the 48% survival in our experiments estimated from the Kaplan analysis at 60 days post infection. The COL population was clearly more resistant to

NHP-B than the KONA SPF population with significantly greater survival (Fig.2). The higher level of resistance of the COL population is consistent with the very low incidence of infection by NHP-B in commercial grow out ponds in Colombia. Most of the countries in the Americas that farm P. vannamei have episodes of NHP. Countries, including Peru,

Ecuador, Venezuela, Brazil, Mexico and most Central American countries, have reported

NHP outbreaks in the juvenile stages causing mortalities range from 50 to 99% (Lightner

1996). Unlike other countries, NHP in Colombia has only been reported in broodstock ponds and just a very few cases has it been found in grow-out ponds (Briñez et al. 2003).

Despite having temperatures and salinity conditions that favor NHP outbreaks, the disease seems to be restricted to sub-adults and broodstock ponds in Colombia.

The resistance of COL population to NHP-B infection might be related to the process of selection for resistance to TSV. During 1992-1995 most of Latin American countries that raised P. vannamei were severely affected by Taura syndrome which caused massive

106 losses in the shrimp industry. Several shrimp breeding programs were initiated at this time to develop TSV resistant populations. In Colombia, the shrimp industry set up a mass selection program in which growers selected surviving shrimp from the highly TSV affected ponds (Cock et al. 2009). The three largest producers in the Caribbean coast began raising survivors from heavily TSV infected ponds. Those farms typically had salinities that ranged from 10-15 ppt in the rainy season to 35-40 ppt in the dry season, and mean annual temperatures that typically ranged from 24°C to 32°C, environmental conditions in which NHP-B may cause disease outbreaks (Frelier et al. 1992; Lightner

1996). Although each farm had its own selection and multiplication protocol, the most common practice consisted of the transfer of survivors from a commercial grow-out pond at about 4 months to a lower population density pond before being transferred to the maturation lab. In each transfer, the larger, healthier shrimp with no deformities, flaccid bodies or melanization were transferred to the next phase. Obviously, no abnormal appearing or moribund shrimp passed from one phase to the next in this broodstock production scheme.

NHP-B has been reported in sub-adults and broodstock populations in the farms where mass selection was carried out with high prevalence in some ponds (Briñez et al. 2003;

Aranguren et al. 2006). Typical NHP clinical signs in symptomatic shrimp are soft shell and flaccid bodies. Thus, a mass selection program ostensibly directed to TSV resistance, but which uses as selection criteria survival and elimination of animals with soft shells and flaccid bodies, in the presence of both TSV and NHP-B will simultaneously select

107 for resistance to both TSV and NHP-B. The selection pressure in the initial grow out phase may have been low due to the low levels of NHP-B encountered in grow out ponds. However, due to the almost continual presence of NHP-B in the broodstock ponds, uninfected shrimp in the initial selection from commercial grow out ponds would likely have been challenged by NHP-B in the maturation stage in the broodstock ponds.

Thus, selection for NHP-B resistance would have probably been enhanced by the continuous exposure to NHP-B which was the main disease present in the broodstock ponds. The resistance obtained in this manner could either be resistance to infection or the ability to survive and grow as asymptomatic carriers. Aranguren et al. (2006) showed that asymptomatic carriers, or animals with limited signs of infection, are capable of spawning, suggesting that selection may have been for resistance in asymptomatic animals or those that show low levels of disease expression. This view is supported by the lack of difference in NHP-B load in the susceptible Kona SPF lines and the resistant

COL populations. In chronic diseases such as NHP, resistance is frequently controlled by many genes (Rolff and Silvajothy, 2003) and selection over several cycles is likely to increase the frequency of those genes in the population providing stable resistance which is unlikely to suddenly to break down with the evolution of new strains of the causal agent. Thus, the unplanned selection over several cycles for resistance to NHP, may explain the very low incidence of NHP disease in grow-out ponds, restricting NHP to those ponds where water quality and other unknown factors favor the expression of NHP-

B infection.

108

TSV resistant lines that have been selected in absence of NHP-B have been used by commercial growers in Central and South America. In those areas, serious NHP-B outbreaks have been reported (Lightner and Redman, submitted; Morales et al. 2006), suggesting that TSV does not confer a generalized resistance to other pathogens including

NHP-B.

In summary, we describe here a real-time method for the diagnosis and quantification of

NHP-B in shrimp HP and feces samples. The assay is specific and sensitive. The sensitivity of the conventional NHP PCR can be increased to a detection limit of 103 copies by increasing the number of PCR cycles to 35. The COL population, selected for several generations in the presence of NHP-B infection, is more resistant to NHP disease than the Kona SPF line. The low incidence of NHP in commercial grow out ponds in

Colombia is likely a result of the resistance of the COL populations.

Acknowledgements

This work was supported by the Gulf Coast Research Laboratory Consortium Marine

Shrimp Farming Program, USDA Grant No. USMSFD 2009 / 19851-UAZ and the S-K grant No: NA09NMF4270102, NOAA. We thank to Dr. Marcela Salazar and Dr. James

Cock for the critical reviewing of this manuscript, Linda Nunan and Brenda Noble for providing technical assistance, Rita Redman for histological services and Dr. H. Harris for providing the NHP-B infected tissue.

109

Tables and Figures

Table 5.1 Reproductibility of the TaqMan real time qPCR in six different assays. SD:

Standard Deviation; Cq: Quantification cycle

Inter-assay SD NHP Intra-assay SD (Average Cq value for duplicate) (Mean Cq copies 1 2 3 4 5 6 value) 1x10 2 1.1 (36.5) 0.6 (36.2) 0.7 (36.4) 0.7 (36.3) 0.0 (35.3) 0.1 (35.8) 0.5 (36.1) 1x10 3 0.2 (32.8) 0.1 (33.2) 0.1 (32.0) 0.8 (33.0) 0.4 (31.6) 3.1 (34.2) 0.8 (32.8) 1x10 4 0.5 (29.9) 0.2 (29.4) 0.2 (29.3) 1.2 (30.1) 1.2 (30.1) 1.3 (29.6) 0.8 (29.7) 1x10 5 0.1 (25.7) 0.4 (26.4) 0.2 (25.6) 0.2 (25.8) 0.3 (25.4) 0.0 (25.1) 0.2 (25.7) 1x10 6 0.1 (22.2) 0.3 (22.5) 0.0 (22.4) 0.3 (22.2) 0.2 (21.0) 0.0 (22.1) 0.2 (22.1) 1x10 7 0.1 (18.9) 0.1 (18.8) 0.1 (19.0) 0.2 (18.7) 0.2 (17.9) 0.3 (18.5) 0.2 (18.6) 1x10 8 0.1 (15.6) 0.2 (15.5) 0.3 (16.1) 0.1 (16.0) 0.2 (15.3) 0.1 (15.6) 0.2 (15.7)

110

Table 5.2 Final survival of two Penaeus vannamei populations infected with NHP-B by reverse gavage.

Tank Control Treatment tanks 1 2 Mean COL 92% 60% 82% 71%* KONA 90% 32% 50% 41%

* Indicates significant differences

111

Table 5.3 Mean ± standard error of NHP-B copy μg-1 of DNA in HP samples of COL and

KONA Penaeus vannamei shrimp populations in tanks 1 and 2 after NHP-B challenge test.

Tank Tank 1 Tank 2 Population Infected shrimp (%) Copies μg-1 Infected shrimp (%) Copies μg-1 (n) of DNA ±SE (n=14) (n) of DNA ±SE (n=11) COL 100.0 (14/14) 1.61x107 ±4.59x106 78.5 (11/14) 1.24x102±2.88x101 KONA 100.0 (14/14) 1.52x107 ±7.57x106 78.5 (11/14) 1.21x102 ±3.14x101

112

Fig. 5.1 Standard curve of the NHP-B copy number versus Cq (Quantification cycle) value. Purified pNHP1 plasmid was serially diluted from 1x108 to 10 copies and used as template in qPCR. (Cycle) Cq

113

Fig 5.2 Cumulative survival of COL and KONA Penaeus vannamei populations challenged with NHP-B. COL (solid square) (n=100) and KONA (solid triangle)

(n=100); Controls (dashed lines) n=50. Mean± SE per duplicate. (p<0.05)

100

25 Cumulative Survival (%)

0 0 7 21 35 63 77 90 116 days

114

Fig.5.3 NHP-B results using conventional NHP PCR.A: 25 cycles, B: 35 cycles. Samples

1: 8.7x101, 2: 8.7x102 3: 8.7x103, 4: 8.7x104 5: 8.7x105 6: 8.7x106 7:8.7x107. ntc: no

template control, +C: Positive control, M: 100 bp ladder.

1 2 3 4 5 6 7 ntc +C M 400 bp A: 25 cycles 300 bp

400 bp B: 35 cycles 300 bp

379 bp

115

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