Aquaculture 229 (2004) 1 – 10

www.elsevier.com/locate/aqua-online

Real-time RT-PCR determination of viral copy

number in Penaeus vannamei experimentally
infected with Taura syndrome virus
L.M. Nunan*, K. Tang-Nelson, D.V. Lightner
Department of Veterinary Science and Microbiology, University of Arizona,
Building 90, Tucson, AZ 85721, USA
Received 17 November 2002; received in revised form 22 April 2003; accepted 25 April 2003

Abstract

This study examined the viral copy number as determined by real-time RT-PCR, in different
tissue samples from Penaeus vannamei exposed to Taura syndrome virus (TSV). Two routes of
exposure, injection and per os, were investigated. Six different body parts from each shrimp were
assessed for viral copy numbers. Eight shrimp were analyzed per treatment. In addition, eight
specific pathogen free (SPF) P. vannamei were analyzed and served as a negative control. The tissue
samples examined included: whole tail muscle, tail muscle (shell removed), gills, pleopods, head
(cephalothorax with the hepatopancreas included) and tail fan. The results from these experiments
showed a significant level of difference between the SPF and the injection treatments. As was
expected, there was also a significant difference between the negative control and the per os
treatment groups. There was no significant difference between the viral copy number contained in
different body parts from the injection experiment. These results contrasted to the per os results, in
which there was a statistically significant difference between tail/gills, tail/head, tail/tail fan, whole
tail/tail fan and pleopods/tail fan. The tail samples had the lower viral copy numbers, as did the
whole tail and pleopods when compared to the tail fan. The mean viral copy number per nanogram of
total RNA (x̄ cn/ng tRNA) extracted in the injection study ranged from 1.4  105 in the gills to
2.3  105 in the whole tail. In the per os experiment, the x̄ cn/ng of extracted tRNA ranged from
2.5  104 in the tail muscle to 4.3  105 in the head. When these values are converted to mean viral
copy number per gram (x̄ cn/g) of tissue, the values increased in range. In the injection study, the x̄
cn/g of tissue ranged from 1.2  109 in the tail fan to 7.4  109 in the head. These values contrast to
the x̄ cn/g of tissue in the per os study in that the lowest value of 1.7  108 was in the tail muscle and

* Corresponding author. Tel.: +1-520-621-4438; fax: +1-520-621-4899.

E-mail address: lmn@u.arizona.edu (L.M. Nunan).

0044-8486/$ - see front matter D 2004 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0044-8486(03)00365-X
2 L.M. Nunan et al. / Aquaculture 229 (2003) 1–10

the highest x̄, 1.7  1010, was in the head. Overall, all body parts from infected shrimp, regardless of
treatment type, quantitatively analyzed by real-time RT-PCR, determined the presence of TSV.
D 2004 Elsevier Science B.V. All rights reserved.

Keywords: TSV; Shrimp; Penaeus; Real-time PCR

1. Introduction

The worldwide shrimp farming industry continues to be adversely affected by

epizootics due to viral pathogens. Taura syndrome virus (TSV) has had an enormous
negative economic impact on Penaeus vannamei shrimp culture in the Western Hemi-
sphere (Lightner, 1996a; Hasson et al., 1999a). Over the past few years, areas in Asia have
begun to culture P. vannamei, and in so doing, have experienced TSV outbreaks (Yu and
Song, 2000).
The spread of exotic pathogens from one geographical region to another can be
attributed to several avenues of introduction. Importation of infected stocks, birds acting as
vectors, ballast water discharge and human sewage are several documented or suggested
methods by which pathogens can be moved to different locations (Schnurrenberger et al.,
1987; Garza et al., 1997; Lightner et al., 2001; Subasinghe and Bernoth, 2000). Another
way by which pathogens can be moved to new geographic locales is through the
importation and reprocessing of frozen commodity products (Nunan et al., 1998; Durand
et al., 2000; Soto et al., 2001).
Exotic pathogen introduction and establishment has been a concern in both the United
States and Australia. To address this concern, each country carried out risk assessments
(RA) posed by the various routes of introduction (AQIS, 2000; Van der Schalie and
Austin, 1998). Unfortunately, the conclusions reached from the U.S. RA were limited due
to the lack of research on this topic. Durand (2003) subsequently studied the quantitative
viral loads in P. vannamei infected with white spot syndrome virus (WSSV), and
determined that total virus load on a per weight basis was similar in the head and tail.
The implications from this study for RA was that reprocessing and packaging of WSSV
infected whole or de-headed shrimp pose a significant hazard of spreading within the
aquatic environment and infecting susceptible, native species if the processing wastes are
improperly disposed of.
The purpose of this study was to determine quantitatively the viral loads of different
parts of the shrimp, P. vannamei, infected with TSV. Using real-time RT-PCR method-
ology, six distinct body parts were analyzed. Two different routes of exposure to TSV were
used to induce the disease and experimentally simulate emergency harvests during the
acute phase of the disease (Hasson et al., 1999b). In 1997, Lotz performed a study that
concluded that shrimp mortality from Taura virus exposure was higher by injection than by
ingestion and that injection results in a more virulent infection. At that time, real-time PCR
was not a procedural option. In the present study, specific pathogen free (SPF) P. vannamei
were infected by either injection or per os treatment and distinct shrimp body parts were
analyzed for viral copy numbers using real-time RT-PCR.
 L.M. Nunan et al. / Aquaculture 229 (2003) 1–10 3

2. Materials and methods

In the injection experiment, 100 specific pathogen free (SPF) 3 g juvenile, P. vannamei
were injected with a diluted TSV homogenate. These shrimp were stocked into a 1000-
l tank containing artificial seawater and maintained on a commercial pelleted ration. The
first mortalities were observed at day 2 post injection (PI). After observing the initial
mortalities, on day 3 PI, the surviving shrimp were collected to simulate an emergency
harvest. Five shrimp were preserved in Davidson’s fixative for TSV confirmation by
standard histology and in situ hybridization assay with a specific TSV DNA probe and the
remainder were frozen at 70 jC for later analysis by real-time RT-PCR. Representative
shrimp sampled on day 0 served as the negative control for in situ hybridization and real-
time RT-PCR assays and confirmed the initial SPF status of the test animals.
In the per os experiment, 100 SPF P. vannamei were fed minced TSV tissue, at a rate of
5% body weight for two consecutive days. Following the feeding of TSV-infected tissue,
the experimental shrimp were maintained on a pelleted ration. After 5 days, the first
mortalities were observed, so 6 days following the first feeding, surviving shrimp were
collected and either frozen at 70 jC or preserved in Davidson’s fixative for analysis by
real-time RT-PCR or standard histology and in situ hybridization, respectively.

2.1. Histology and in situ hybridization

Five shrimp were randomly selected from the simulated emergency harvests and
preserved in Davidson’s fixative. The specimens were processed for routine histology
using standard methods (Lightner, 1996b). Two SPF shrimp (day 0) were sampled and these
served as the negative controls for both histological and in situ hybridization analysis.
Tissue samples were embedded in ParaPlast Plus paraffin (Fisher Scientific, Pittsburg, PA)
and sectioned at 4-Am thickness, mounted onto microscope slides, deparaffinized and
stained with haematoxylin and eosin (H&E) (Lightner, 1996b). Histological examination of
the processed samples was performed using standard light microscopy. For in situ
hybridization assays, the paraffin embedded tissues were mounted onto positively charged
slides and hybridized with a TSV-specific digoxigenin-labeled probe (Mari et al., 1998).

2.2. Real-time RT-PCR quantification of TSV

Real-time RT-PCR was used to determine the viral copy number in six distinct tissue
body part samples from a single shrimp. The tissues examined included: whole tail muscle
(shell included), tail muscle, gills, pleopods, head (cephalothorax with the hepatopancreas
included), and tail fan (telson and uropods) (Fig. 1). Eight shrimp from the injection, per os
and SPF treatment groups were analyzed. Each body part was run in triplicate in the real-
time RT-PCR format.

2.3. RNA extraction

From the experimental samples that had been frozen at 70 jC following the
emergency harvests, total shrimp RNA was extracted from 25 to 50 mg of tissue using
4 L.M. Nunan et al. / Aquaculture 229 (2003) 1–10

Fig. 1. Mean TSV copy number per nanogram of extracted RNA in different body parts of juvenile P. vannamei
from a simulated emergency harvest (I = injection, PO = per os exposure).

the High Pure RNA Tissue kit (Roche Molecular Biochemical, Indianapolis, IN, USA) by
following the manufacturer’s protocol. The concentration of total RNA in each sample was
estimated using a BioPhotometer spectrophotometer (Eppendorf Scientific, Hamburg,
Germany).

2.4. Primers and probe

The RT-PCR primers and TaqMan probe for the detection of TSV were selected from
ORF1 region of the TSV genomic sequence (Mari et al., 2002, GenBank AF277675). The
primers and probe sequences were designed with Primer Express software (Applied
Biosystems, Foster City, CA, USA). The primers (TSV1004F/TSV1075R) generate a 72-
bp amplicon. The TaqMan probe (TSV-P1) which corresponds to the region from
nucleotide 1024 to 1051, was synthesized and labeled with fluorescent dyes 6-carboxy-
fluorescein (FAM) at the 5Vend and 6-carboxy-N,N,NV,NV-tetramethylrhodamine (TAMRA) at
the 3Vend (Table 1).

2.5. Real-time RT-PCR reagents and cycling

The assays were performed on an GeneAmp 5700 sequence detection system with
TaqMan One-Step RT-PCR master mixture (Applied Biosystems) containing: Multi-
Scribek (a reverse transcriptase), RNA inhibitor, AmpliTaq Gold DNA polymerase,

Table 1
The sequences of TSV primers and TaqMan probe
Upstream primer (TSV1004F)
Downstream primer (TSV1075R)
TaqMan probe (TSV-P1)
5V-TTG GGC ACC AAA CGA CAT T-3V
5V-GGG AGC TTA AAC TGG ACA CAC TGT-3V
5V-CAG CAC TGA CGC ACA ATA TTC GAG CAT C-3V
 L.M. Nunan et al. / Aquaculture 229 (2003) 1–10 5

dNTPs with dUTP, Passive Reference. A sample of 20 ng of extracted RNA was added to a
RT-PCR mixture containing 0.3 AM of each primer and 0.2 AM of TaqMan probe in a final
volume of 25 Al. Amplification was performed with the following cycling profile: 30 min
reaction for reverse transcription at 48 jC and subsequent activation of the AmpliTaq Gold
for 10 min at 95 jC, followed by 40 cycles of 15 s at 95 jC and 1 min at 60 jC. The viral
quantity of each sample was determined by GeneAmp 5700 Sequence Detection System
software (SDS 1.0). Each sample was tested in triplicate. A series of dilutions from a TSV
recombinant plasmid in which the copy number was known, were used as the positive
control and standards for quantification in all real-time assays.
Construction and cloning of the positive control plasmid was accomplished through the
use of a PCR fragment containing 416 bp of TSV genomic sequence (nucleotide 837 –
1252) that was amplified by RT-PCR from purified TSV virions. This fragment was
purified with a PCR purification kit (Qiagen, CA, USA) and ligated to pGEM-T easy
vector (Promega, WI, USA). The positive clone, pTSV-1, was verified by DNA
sequencing. To prepare the RNA standard, the pTSV-1 plasmid was cloned in Escherichia
coli JM109 competent cells, purified with a High Pure Plasmid Isolation Kit (Roche
Molecular Biochemicals), and linearized by PstI digestion. The linearized plasmid was
used as the template for in vitro transcription with T7 MAXIScript (Ambion, TX, USA).
The RNA was extracted by phenol – chloroform, precipitated with ethanol and quantitated
with a spectrophotometer.
The mean and standard deviation (S.D.) of the three replicate real-time RT-PCR values
were determined and values that did not deviate by more than 1 S.D. from the mean were
considered valid data. Outliers were not included in the data analysis. A one-way analysis
of variance (ANOVA) test (Nelder and Wedderburn, 1972; Ott, 1993) was used to compare
the means of the TSV viral copy number in the treatment groups (SPF, injection and per
os). A t-test (Ott, 1993) was used to compare the means among the TSV copy numbers
generated from the real-time RT-PCR assay triplicate values from the six different body
parts of the shrimp. All data was transformed to log base 10 for ease of handling and data
was analyzed as a completely randomized design. Alpha was set to 0.05. From the
resulting statistics, the 95% confidence interval (CI) for the comparison of shrimp parts by
treatment was derived.

3. Results

Experimental shrimp in the injection study began to die 2-day PI. In the per os study,
the shrimp first showed mortalities at 5-day PI. Simulated emergency harvests occurred on
the day following the onset of mortalities (injection 3-days and per os 6-days).

3.1. Histological and in situ hybridization results

The results from the histological sections processed using routine H&E staining and in
situ hybridization assays with a TSV-specific probe confirmed the infections in both the
per os and injection treatment groups. Histological evaluation of the 10 samples from the
injection and per os bioassays revealed a range of infection from not detected through
6 L.M. Nunan et al. / Aquaculture 229 (2003) 1–10

severe TSV infection (Lightner, 1996b). From the samples in both the injection and per os
treatments that displayed lesions, the target tissues were the cuticular epithelium of the
carapace, stomach, body appendages and gills, which were characterized by necrosis and
nuclear pyknosis of the cells. Within the lesions were multiple basophilic spheres that
represent intracytoplasmic inclusion bodies, pyknotic and karyorrhectic nuclei. The in situ
assays showed a strong hybridization signal in the cytoplasm of the target tissues and in
the lesions delineated by histology (Fig. 2). Histological examination and in situ
hybridization assays of the 0-day samples and the SPF control group confirmed the initial
uninfected status of the experimental shrimp used in all of the bioassays.

Fig. 2. In situ hybridization assay results from the cuticular epithelium in the stomach of P. vannamei infected
with TSV by (A) injection and (B) per os. The infected areas reacted strongly to the TSV-specific probe as is
visualized by the black precipitate (arrows). Normal cuticular epithelial cells do not show any hybridization
reaction to the probe. Bars = 20 Am.
 L.M. Nunan et al. / Aquaculture 229 (2003) 1–10 7

Table 2
Mean values of total RNA after extraction from shrimp tissues
Tissue Extracted total RNA (ng of RNA/mg of tissue) Total RNA yield (ng of RNA)
Injection Per os SPF Injection Per os SPF
Whole tail 10.1 9.6 9.4 666.3 565.0 548.8
Tail muscle 11.7 8.4 7.6 721.3 512.5 473.8
Gills 20.8 11.2 9.2 1068.8 677.5 597.5
Pleopods 10.8 12.1 7.5 640.0 655.0 527.5
Head 149.9 56.9 21.6 11,207.5 4048.8 1575.0
Tail fan 8.7 11.1 7.0 787.5 693.8 487.5

3.2. TSV quantification by real-time RT-PCR

The RNA yield from the various tissues, following extraction with the High Pure RNA
Tissue Kit, was generally consistent with the expected range of 300 –3000 ng of total RNA
as specified by the manufacturer (Table 2). The exception in the values was seen in the
head tissue in which the total RNA extracted exceeded the expected range in both the
injection and per os treatments, but not in the SPF control. The variability in RNA yield
between body parts was compensated for by using 20 ng of extracted RNA from each
sample that was tested in the real-time RT-PCR format.
In each real-time run, a standard curve was generated from the purified TSV RNA that
ranged between 100 and 1.00  109 copies. Copy numbers were calculated by interpolat-
ing the experimentally determined threshold cycle (Ct). From this data, the results were
summarized as mean copy number of TSV per nanogram of total RNA (x̄ cn/ng tRNA)
and mean copy number of TSV per gram (x̄ cn/g) of tissue (Table 3).

Table 3
Mean values of copy number per nanogram of total TSV RNA and per gram of tissue of total TSV RNA from
whole tail, tail muscle, gills, pleopods, head, and tail fan samples of P. vannamei with acute TSV infections in
injection and per os experiments
Treatment Sample N Copy number/nanogram of total Copy number/gram of tissue
extracted RNA
Mean Range Mean Range
Injection Whole tail 8 2.3  105 3.5  103 – 8.9  105 1.7  109 6.5  107 – 4.3  109
Per os 5.6  104 3.4  103 – 1.7  105 5.4  108 2.1  107 – 1.2  1010
Injection Tail muscle 8 2.0  105 3.1  103 – 9.5  105 1.9  109 6.0  107 – 9.6  1010
Per os 2.5  104 1.7  103 – 1.1  105 1.7  108 9.6  106 – 6.5  108
Injection Gills 8 1.4  105 1.5  104 – 5.5  105 1.7  109 2.8  108 – 5.3  109
Per os 7.6  104 7.5  103 – 3.5  105 6.4  108 6.0  107 – 1.2  109
Injection Pleopods 8 1.5  105 1.2  104 – 5.8  105 1.3  109 1.4  108 – 4.9  109
Per os 6.8  104 1.5  103 – 4.3  105 5.1  108 4.2  107 – 3.0  109
Injection Head 8 1.9  105 4.5  103 – 9.0  105 7.4  109 8.7  108 – 2.5  1010
Per os 4.3  105 1.8  103 – 2.5  106 1.7  1010 3.4  107 – 9.2  1010
Injection Tail fan 8 2.1  105 1.9  104 – 6.1  105 1.2  109 5.0  108 – 2.8  109
Per os 1.2  105 5.4  103 – 6.5  105 8.2  108 5.6  107 – 4.3  109
8 L.M. Nunan et al. / Aquaculture 229 (2003) 1–10

Table 4
Comparison of p values from the injection and per os bioassays using a two-sample t-test in which significance is
p < 0.05 at a 95% confidence interval (significant differences are denoted in bold print)
Treatment Shrimp body parts Tail muscle Gills Pleopods Head Tail fan
Injection Whole tail 0.26 0.42 0.34 0.30 0.75
Per os 0.058 0.45 0.12 0.12 0.031
Injection Tail muscle 0.97 0.92 0.65 0.31
Per os 0.008 0.43 0.035 0.017
Injection Gills 0.92 0.77 0.23
Per os 0.071 0.60 0.71
Injection Pleopods 0.83 0.17
Per os 0.081 0.005
Injection Head 0.16
Per os 0.62

Analysis of the x̄ values and standard deviation (S.D.) of the triplicate RT-PCR values
determined the integrity of the data. In each triplicate, values that did not deviate by more
than 1 S.D. from the mean were considered valid data. Data from the injection treatment
had four values with S.D. of 1.02, 1.43, 1.88 and 1.48. Two outliers were from the whole
tail (different triplicates), one from the gills and one from the tail fan. The per os treatment
data revealed only one outlier in one triplicate set from the tail fan data that had a S.D. of
1.22.
The x̄ cn/g of tissue from the injection bioassay ranged from 1.2  109 in the tail fan to
7.4  109 in the head. These values contrasted the per os bioassay values which ranged
from 1.7  108 x̄ cn/g in the tail muscle to 1.7  1010 x̄ cn/g in the head. The x̄ cn/g of
tissue from the injection bioassay were greater in all body parts, except the head, when
compared to the values generated from the per os bioassay.
Using the triplicate values (outliers removed) generated by real-time RT-PCR, treatment
types (injection, per os, and SPF) were compared using one-way analysis of variance
(ANOVA) and as was expected, determined a significant difference between injection and
SPF treatments and per os and SPF treatments ( p < 0.0001). There was no significant
difference ( p < 0.05) in the x̄ cn in the injection bioassay when tissues were compared
using two-sample t-test . The results from the per os bioassay that compared viral load
among body parts showed a significant level of difference ( p < 0.05) in the x̄ cn between
the tail/gills, tail/head, tail/tail fan, whole tail/tail fan and the pleopods/tail fan. The tail
samples had the significantly lower viral x̄ cn, as did the whole tail and pleopods, when
compared to the tail fan (Table 4).

4. Discussion

Routine histology and in situ hybridization assays confirmed the TSV infections from
the injection and per os bioassays. The initial SPF status of the experimental shrimp was
also confirmed by these techniques. Real-time RT-PCR was used to quantitatively
establish the viral loads in distinct body parts in the P. vannamei from the simulated
emergency harvests.
 L.M. Nunan et al. / Aquaculture 229 (2003) 1–10 9

In the injection study, mean copy number per gram (x̄ cn/g) of tissue consistently was in
the 109 range for all shrimp parts analyzed. The per os treatment showed that the x̄ cn/g of
tissue ranged between 108 and 1010. The viral x̄ cn/g in the head in both the injection and
per os treatments contained the greatest load of virus. Many of the target tissues of TSV,
including the stomach and cuticular epithelium of the carapace, are contained in the head,
and this may explain the higher concentration of the virus in these samples. From the data
generated, comparisons can be made between the viral loads contained in heads and tails.
In both injection and per os exposures, the heads (gills included) contained more virus than
the tails (whole tail, tail muscle, pleopods and tail fan).
Further research using the quantitative abilities of real time RT-PCR should be conducted
on the amount of virus required to induce disease. From the data presented in this study, TSV
was determined to be present within all the shrimp body parts assessed. Future research
should determine if the viral loads within the parts of the shrimp exceed some minimum
threshold needed to be infectious by cannibalism or as measured experimentally through per
os challenges. In addition, using the real-time RT-PCR technique, infectivity studies should
be conducted to ascertain the relationship between initial viral copy number of the inoculum,
either homogenate for injection or tissue for per os exposure, related to viral copy numbers
determined in the infected shrimp. This information may allow for direct comparison
between the virulence of TSV in the two types of exposure.
With the introduction of P. vannamei into areas of Asia and the subsequent appearance
of TSV, the threat posed by reprocessing and packaging commodity shrimp increases the
risk of the spread of this disease. Body parts that can be discarded during the processing or
value added reprocessing of shrimp may include the head, gills, pleopods, shell and the tail
fan. As was demonstrated in this study, all of these tissues contained the virus, in fact, the
head was shown to have the greatest viral loads. Therefore, processing shrimp can pose a
threat of contaminating the aquatic environment if infected waste parts are improperly
disposed of in coastal regions. Through the quantification of viral copy numbers in specific
body parts of the shrimp, more informed Risk Assessments can be performed to determine
the potential danger and spread of the virus to naı̈ve animals, from disposal of shrimp waste
parts into the aquatic environment.

Acknowledgements

Funding for this research was provided by a grant from the USMSFP, USDA, CREES,
Grant No. 99-38808-7431. The authors would like to thank Brenda White-Noble, Dr.
Carlos Pantoja, Leone Mohney, and Rita Redman for their input and/or assistance in
conducting these experiments and Doug Jones and Wayne Nicholson for their advice on
evaluation of the statistical data.

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