魚 病 研 究 Fish Pathology, 36 (3), 139-146, 2001.9 2001 The Japanese Society of Fish Pathology

Detection of Infections Pancreatic Necrosis Virus (IPNV) from Leukocytes of Carrier Rainbow Trout

Oncorhynchus mykiss

Sylvia Rodriguez, Marta Alonso and Sara I. Perez-Prieto*

Centro de Investigaciones Biologicas, Departamento de Microbiologia Molecular, C/Velazquez 144, 28006 Madrid, Spain

(Received February 16, 2001)

ABSTRACT--Blood and kidney leukocytes were purified from two-year-old rainbow trout carrying infectious pancreatic necrosis virus (IPNV) and the cells were processed to detect the virus by two methods: 1) indirect immunofluorescence stain followed by flow cytometry analysis, and 2) RT-PCR or nested-PCR amplification. These methods were compared with separate homogenization of visceral samples, followed by inoculation on cell cultures and seroneutralization, a procedure routinely employed by most disease diagnostic laboratories. IPNV was isolated by homogenization of samples in all seven specimens examined. The assay took between 7 and 28 days required for the appearance of cytopathic effects, which is usual in subclinical samples. From

purified leukocytes, IPNV was detected by RT-PCR in five out of the seven specimens. The nested-PCR improved the sensitivity of the assay and gave positive results for all the fish. In addition, flow cytometry demonstrated the presence of IPNV in all the fish in 8-24 h. Examination of leukocytes is a practical way of detecting IPNV and much less time-consuming than the homogenization technique.

Key words: IPNV, PCR, flow cytometry, leukocyte, carrier, detection, Oncorhynchus mykiss

Infectious pancreatic necrosis virus (IPNV) is an economically important fish pathogen which causes severe acute infections in young salmonid fish, resulting in high mortality. The virus is the type species of the family Birnaviridae and may also infect different species of teleost fish in both natural and artificial environments

(Wolf, 1988). Following a disease outbreak, surviving fish may become virus carriers without showing symptoms. The presence of carrier fish leads to epizootics in

younger fish (Yamamoto, 1975; Magunwiryo and Agius, 1988). Control of IPNV by vaccination has not yet been achieved, so most hatchery efforts to date have been attempted to eliminate the virus during juvenile development. An integral part of avoiding juvenile disease has been to select broodstock which have never encountered the virus. The diagnosis of infection is usually based on the isolation of the virus from visceral samples in cell culture and identification of the pathogen by

seroneutralization or immunofluorescence test (Amos,

1985). Several authors have described PCR assays for the identification of specific strains of IPNV in infected cell cultures that can improve the diagnosis of the virus.

(Heppell et al., 1992; McAllister et al., 1991; Lopez-Lastra et al., 1994; Blake et al., 1995; Pryde et al., 1993).

However, isolation of IPNV from asymptomatic populations of adult fish, in which the presence of defective viruses or low levels of infectious virus are usual, is more difficult than isolation from diseased fish.

Here we attempt to improve results in the detection of IPNV from fish with subclinical infection, using leukocytes as the substrate and RT-PCR and flow cytometry assays. Since leukotropism of IPNV has been repeatedly described (Sadasiv, 1995), these cells may carry IPNV for long periods.

Material and Methods

Cells and virus The BF-2 cell line from bluegill Lepomis macrochirus

fry (American Type Culture Collection, ATCC CCL 91) was used for the propagation, quantification and identifi-

* Corresponding author

E-mail: saraip@cib.csic.es

140S. Rodriguez, M. Alonso and S. I. Perez-Prieto

cation of the viruses. Cells were propagated in

Leibovitz growth medium (L15, Gibco) supplemented

with 10% fetal bovine serum, 100 IU penicillin/mL and

100 mg streptomycin/mL. The maintenance medium

(MM) was the same, except that the serum concentra

tion was reduced to 2%. The BF-2 cell line was incu

bated at 25•Ž for routine cell propagation.

The strain IPNV Sp (ATCC VR 1318), was cultured

at 19•Ž and used as reference virus. The virus was

propagated on BF2-cells and the infective titer was de

termined by the 50% tissue culture infective dose

(TCID50/mL).

Fish sampling and virus isolation

In 1997 specimens were taken from a farm where

only rainbow trout (Oncorhynchus mykiss, Richardson)

were raised. The farm had no history of virus infection

until a batch of 200 adult fish (800g, two years old) was

introduced in 1996. Their progeny developed symp

toms of IPNV disease at 3-4 mo old, and 30 of these

(3-4 cm) were sampled and processed for virus detec

tion following standard protocols (Amos, 1985).

Because IPNV was isolated, the broodstock was

suspected to be IPNV carrier and seven fish were se

lected for confirmation.

Blood, spleen, liver, pancreas pyloric-caeca, kidney,

gonads and brain were aseptically extracted. Blood

was collected in heparinized tubes and diluted (1:5) in

Hanks' saline buffer. The samples were sent refriger

ated (4•Ž) by express mail from the farm to the labora

tory (24 h). Each visceral sample was then separately

tested by the homogenization method. Leukocytes

were collected from blood and kidney. Cells from the

pronephros were obtained by pressing the tissue

through a stainless-steel mesh in cold serum free

medium. Leukocytes were isolated through a Ficoll

-Paque (Pharmacia Biotech Uppsala Sweden). The di

luted blood or kidney homogenates were carefully lay

ered on top of 3 mL Ficoll-Paque in sterile plastic tubes.

The tubes were centrifuged at 300•~g for 30 min at 10•Ž

and the thin band of leukocytes located at the plasma/

ficoll interface was carefully aspirated with a capillary

pipette. Samples contaminated with erythrocytes dur

ing this operation were processed again. Leukocyte

samples were washed in Hanks' saline, centrifuged and

resuspended in phosphate-buffered saline

Flow cytometry

The flow cytometry requires analysis of individual

cells and the previous determination of the specificity of

the method for each cell population. Appropriate

reagents and a negative cell population must provide the

control histogram in which the greatest number of cells

have very low fluorescence levels, allowing determina

tion of the specificity threshold. This is showed by a

dotted line in the graph that represents the fluorescence

intensity channel number above which the cells would be considered as positive. In the histogram, the negative cells are situated at the left of the dotted line and the

positive cells at the right; therefore, an adequate control histogram would show near of 95% cells at the left and no more than 5% at the right. This overlap of true

positive and negative cells represents the background fluorescence level of the analysis, being considered

suitable a 5% when using polyclonal antisera. After analysis of the negative control, the number of fluorescent-positive cells which are above of the threshold is automatically recorded and expressed as percentage of the total cell number.

In our experiments, fish from a farm periodically checked by us for virus and considered IPNV-free, were used as negative controls. Leukocyte samples analyzed by conventional and new methods were always negative.

Leukocyte pellets were rinsed once in PBS before fixation with 3.7% formaldehyde in PBS for 15 min; the cells were then washed twice in PBS and permeabilized with 0.01% Triton x-100 in PBS for 1 min.

Drops of anti-IPNV rabbit serum (Vilas et al., 1990; Hill et al., 1981) were added to the pelleted cells, which were gently resuspended and incubated for 30 min at room temperature (Rt). After several washes with PBS+2% FCS, goat anti-rabbit IgG-FITC conjugate

(Sigma) was added and the mixture was incubated again for 30 min at Rt. Tubes were then centrifuged twice in PBS+2% FCS to eliminate non-specific binding, and the fluorescence of the cell suspensions was determined by flow cytometry in an EPICS XL (Coulter, Mostoles, Spain) equipped with an argon ion laser (200 mW at 488 nm excitation). Values of 5000 cells were recorded by sample.

Oligonucleotide primers Two oligonucleotide primers (S1/S2) designed on

the basis of the nucleotide sequence of the IPNV VP-2

gene (Havarstein et al., 1990) were synthesized at the Laboratorio de Quimica de Proteinas, Centro de Investigaciones Biologicas (CSIC), Spain. These primers specifically amplified a 1180-base pair (bp) region

(nucleotides 162-1342) of the VP2 gene. The first primer, S1 (5'-TGAAATCCATTATGCT-TCCAGA

), was 22 nucleotides long and hybridized to positions 162 to 184 (sense orientation) in the open reading frame (ORF) of the VP2 gene; the second, S2

(3'-GACAGGATCATCTTGGCATAGT), was 22 nucleotides long and hybridized to positions 1319-1321 of the ORF (antisense orientation).

To increase the sensitivity of PCR for the detection of virus at low titer, we designed a Nested PCR (NPCR). We selected a set of internal primers V1/V2,

(Pryde et al., 1993) for the amplification of a 613 bp product.

IPNV detection from leukocytes of carrier fish141

The inner primer V1 (5'-GAACCCCCAGGACAA-AGT-3'

) was 18 nucleotides long and hybridized to positions 568 to 585 (sense orientation) in the ORF of the VP2 gene; the second, V2 (5'-TGATTGGTCTGAGCA-CGC-3'

) was 18 nucleotides long and hybridized to positions 1164-1181 of the ORF (antisense orientation).

PCR

Total RNA was extracted from leukocyte pellets of

around 2•~109 cells following the guanidinium thiocyan

ate-phenol-chloroform method (Chomczynski and

Sacchi, 1987). The RNA pellets were washed with 70%

ethanol, dried and resuspended in diethyl pyrocarbonate

treated water. Single stranded c-DNA was synthesized

from 5ƒÊg of total RNA using the first-strand synthesis

kit for RT-PCR as described by the manufacturer

(Amersham Pharmacia Biotech, Essex, UK). Briefly,

RNA was incubated at 42•Ž for 1 h in reverse transcrip

tion reaction mixture (50mM Tris-HCl pH 8.3, 50 mM

KCl, 10 mM MgCl2, 80 mM Sodium pyrophosphate,

10mM each dATP, dGTP, dTTP and 5mM dCTP, 20

units of human placental ribonuclease inhibitor, 70ƒÊM of

random hexanucleotide primer and 5 U of reverse

transcriptase). RNA-DNA hybrids were denatured at

100•Ž for 5 min, and volumes of 2ƒÊL of this reaction

were used for PCR.

Amplification of c-DNA was carried out under the fol

lowing conditions: A mixture containing 2.5 mM MgCl2,

10 mM Tris-HCl pH 8, 50 mM KCl, 50 pmol of each of the

51 and S2 primers, 2 mM deoxinucleoside triphosphate

mix dNTPs and IU Taq-polymerase was added to 2ƒÊL

of the c-DNA template in a final volume of 100ƒÊL.

Nucleic acids from leukocytes of uninfected fish were

used as negative controls. Amplification was per

formed in an automatic thermal cycler (Perkin Elmer

2400) programmed as follows: 3 min at 94•Ž, 35 cycles

of 1 min at 94•Ž, 1 min at 58•Ž, and 1 min at 72•Ž; and

then an extension step at 72•Ž for 9 min. The amplified

products were analyzed by electrophoresis in a 1.2%

agarose gel stained with ethidium bromide.

Nested PCR

Nested PCR (N-PCR) amplifications were carried

out using the RT-PCR products and the inner primer sets

V1-V2. A single ƒÊL of each RT-PCR product was

added to a mixture of 10 mM Tris-HCI pH 8.3, 50 mM

KCl, 2.5 mM MgCl2, 2 mM of each deoxynucleoside

triphosphate, 6 pmol of each primer and 0.4 U of Taq

polymerase, in a final volume of 20ƒÊL. Amplification

involved 30 cycles (94•Ž, 3 min, 58•Ž, 1 min, 72•Ž

1 min) and a final elongation step of 10 min at 72•Ž. N

PCR products were visualized on 1% agarose gel

stained with ethidium bromide.

RESULTS

Recovery of IPNV in cell cultures

Monolayers of BF-2 cells infected with visceral

homogenates from the diseased progeny showed similar

cytopathic effects (CPE) to those of IPNV after 24 or 48

h of incubation at 20•Ž. Seroneutralization confirmed

the presence of IPNV. (Table 1, lane1 ).

The results of virus detection in the seven speci

mens sampled from the broodstock (suspected to be

IPNV carriers) are summarised in Table 2. All the fish

tested were positive for IPNV. The highest isolation

rate was obtained from the spleen, followed by the

gonad and kidney. No virus was detected in the liver or

pyloric caeca, and only one fish was positive for brain

tissue. The level of infective virus was low in all

Table 1. Isolation in BF-2 cells and identification by

seroneutralization and RT-PCR of IPNV from two samples of 30

young rainbow trout

Table 2. Recovery of IPNV in BF-2 cells from individual organs of seven rainbow trout from a 2 years old population. Numbers express the post-infection day in which cytopathic effects were visible.(+, positive CPE; 0, no virus recovery). Blind passages were performed each week. Viral identity was confirmed by seroneutralization

142S. Rodriguez, M. Alonso and S. I. Perez-Prieto

samples and only the spleen samples of specimens 6 and 7 showed early CPE, on the 7th day post-infection. In other tissues, the first CPE were not detected until after at least two or three passages of viral amplification

Homogenate samples from fish of the IPNV-free

population were also tested in cell cultures for verification, being negative (data not shown).

Detection of IPNV by flow cytometry

For determination of the variance of background

Fig. 1. Flow cytometry histograms for samples of leukocytes

from ten fish of an IPNV-free population.

The abscissa scale is arbitrarily divided into 256 channels. The relative frequency of cells with the corresponding fluorescence intensity to the total cell number is expressed on the ordinate scale. The cells were stained by indirect immunofluorescence with a polyclonal anti-IPNV rabbit antiserum and a commercial FITC.anti rabbit IgG, and used to analyse background of fluorescence in healthy fish. The negative cells have very low fluorescence intensities and the intensity channel number under which are situated the

greater part of the cells (around 95%) represents the threshold (dotted line) above which the analysis would be considered positive. The cytometer records automatically the number of this cells , that is expressed as

percentage of positive fluorescent cells, (right angle). A background of 5% (negative cells that overlap with positive cells) is considered suitable for

polyclonal antisera. The histograms from different fish showed fluorescent cell numbers minor than 5%, varying from,1 % to 3%. Values of 5000 cells were recorded by sample. Leukocyte samples from this population were needed as negative control to determine the specificity threshold of fluorescence in each one of the flow cytometer analysis of the IPNV carrier fish.

fluorescence levels in the fish population used as negative control , leukocytes from ten fish individually

sampled and from several blood pools were stained with both anti-IPNV antiserum and IgG-FITC conjugate, and analyzed by flow cytometry. All the histograms showed adequate numbers of fluorescent cells, from 1 to 3 (Fig. 1) or 3.25% (Fig. 3). Similar results were obtained with kidney leukocytes (not shown).

Purified leukocytes from kidney and blood samples of each of the 7 fish were analyzed by flow cytometry

(Figs. 2 and 3) and RT-PCR assay for IPNV detection. Histograms represents quantitative analysis of fluorescence in leukocyte populations stained by IFA with specific IPNV antiserum. The background of fluorescence was less than 3% cells non-specifically stained, and it was measured by staining a negative control, as described in Material and Methods. The dotted line represents the fluorescence intensity channel number above which the cells are considered positive. The percentage of fluorescent cells in the samples is indicated on the graphs and represents percentage of virus-carriers cells or cells expressing viral antigens.

Figure 2 shows the fluorescence records in the

purified leukocytes from the peripheral blood of each adult fish. The negative controls show low background fluorescence and all the samples were clearly positive, varying from 30 to 66 percent of fluorescent cells, consistent with an IPNV carrier state.

Similar results (Fig. 3) were obtained with leukocytes from the anterior kidney of the same specimens. Each fish showed high percentages of cells positive for IPNV. Leukocyte samples from fish 1, 2 and 6 (L1, L6, and L2 histograms) showed more fluorescent kidney leukocytes than those recorded in peripheral blood samples.

Detection of IPNV by RT-PCR and Nested PCR Confirmation of the viral carrier state of the

broodstock was also obtained by using purified leukocytes from kidneys as substrate to RNA extraction for RT-PCR. The oligonucleotide primers S1/S2 directed the synthesis of a 1180 bp segment of DNA from 5 of the 7 RNA leukocyte samples studied. After 30 cycles of PCR amplification, a single band of DNA of the expected molecular weight was observed in samples from fishes 1, 4, 5, 6 and 7 after agarose gel electrophoresis and ethidium bromide staining (Fig. 4A).

N-PCR was then performed, as the amount of DNA

generated by RT-PCR in the presence of low numbers of target molecules was insufficient to produce a visible band in fish 2 and 3. The expected 613 bp product was obtained from each of the 7 samples tested (Fig. 4B). No PCR products were amplified from leukocyte samples of the IPNV-free fish used as negative control

(data not shown). Using pellets of 109 leukocytes for RNA extraction,

IPNV detection from leukocytes of carrier fish143

Fig. 2 Flow cytometry analysis of blood leukocytes from a rainbow trout broodstock carrier of IPNV. The cells were stained by indirect immunofluorescence with a polyclonal anti-IPNV rabbit serum and a commercial FITC-anti rabbit IgG. The abcissa scale is arbitrarily divided into 256 channels. The relative frequency of cells with the corresponding fluorescence intensity to the total cell number is expressed on the ordinate scale. Uninfected control cells (a leukocyte sample from a virus-free

population) were included in the experiment to allow determination of the specificity threshold shown by dotted line. The cytometer records automatically the number of cells above the threshold, that is expressed as percentage of positive fluorescent cells, (the relative number of cells carrying IPNV) on the right angle.

the N-PCR assay was accurate for IPNV detection.

Discussion

The present study shows that detection of IPNV in

asymptomatic adult fish can be more rapid and sensitive

in purified leukocyte samples from kidney and whole

blood than in samples from other organs. According to

table 2, the 7 fish tested were found to be positive for

IPNV by conventional kidney and spleen sampling, but

the time necessary to complete the assay was at least

1-3 weeks. A delay in the appearance of the first CPE

is usual when the viral concentration is low, and some

blind passages are needed to increase the yield, in addition to seroneutralization test to confirm viral identity.

The leukotropism of IPNV permits simple concentration of virus by purification of the cells, which can then we used in flow cytometry analysis or RT-PCR amplification.

Association of IPNV with blood leukocytes from carrier trout has been repeatedly found (Sadasiv,1995), although its ability to replicate in them would appear limited. Detection of IPNV by co-cultivation of leukocytes with a susceptible cell line has been reported by several authors (Swanson and Gillespie, 1982; Yu et al., 1982; Ahne and Thomson, 1986). Agius et al. (1982)

144 S. Rodrlguez, M. Alonso and S. I. Perez-Prieto

Fig. 3. Flow cytometry histograms for IFA stained samples of kidney leukocytes from seven rainbow trout carrier of IPNV. Predefined background of fluorescence was delimited by the negative control(stained leukocytes from an IPNV-free broodstock) and was about 3%. Percentage of fluorescent cells (right angle) express the relative number of cells carrying IPNV.

Fig. 4. Analysis of RT-PCR products and nested-PCR products from leukocyte samples of seven IPNV-carrier rainbow trout. A first step of RT-PCR amplification rendered a 1180 bp product (3A) and asecond step with inner prim-ers rendered a fragment of 613 bp (3B). Lanes (0), DNA ladder molecular weight standard, fragment sizes (inbp) are indicated on the left side of the panel; (1 to 7) RNA extracted from purified leukocytes from fish 1 to 7.

IPNV detection from leukocytes of carrier fish145

also described co-cultivation assays of head kidney cells isolated from IPNV survivors, on RTG-2 cell line, as a sensitive method to detect carriers. In these cells, detection of IPNV by indirect immunofluorescence assay

(IFAT) is tedious and time consuming because a high number of slides must be examined to see relatively few

positive leukocytes. However, techniques such as flow cytometry provides at present the rapid multi-parameter examination of individual cells and offers great potential for investigations in the area of fish pathology

(Chilmonczyk and Monge, 1999; Perez et al., 1994) Earlier studies (Rodrlguez et al., 1991; 1993; 1995),

showed that flow cytometry can be used in the analysis of IPNV in cells and fish; the experiments with leukocytes in vitro, suggested multiplication of IPNV in these cells. In 1995 Johansen and Sommer confirmed the multiplication of IPNV in pronephros adherent leukocytes from Atlantic salmon and suggested a major role for

adherent leukocytes (macrophages) in maintaining the carrier state in salmonid fish.

For a virus to persist undetected, it would either have to be sequestered and present in such low numbers that current systems do not detected it; or be

present only as genetic material which would not interact with the immune system. For diagnostic purposes,

persisting virus is difficult to detect by usual procedures, but the methods described here increase the limit of detection. Flow cytometry requires the analysis of free cells in suspension , and purified leukocyte samples are easy to handle and to stain by IFA, thus providing rapid information.

Low levels of genetic material can be amplified by RT-PCR or nested -PCR assay, which is now used in a variety of viral epizootics. Several reports have indicated that PCR assays can be used to detect and identify specific strains of aquatic birnaviruses in infected cell cultures (Lopez Lastra et al., 1994; Wang et al., 1997; Alonso et al., 1999), but their ability to detect virus from visceral samples in a population of asymptomatic carrier fish is limited (Blake et al., 1995). In the present study,

primers S1/S2 identified IPNV in five of the seven fish. However, in samples with low infective titres, a second step of amplification with primers V1/V2 render a 613 bp fragment, that was necessary to optimize detection. This nested-PCR also confirm the specificity of the 1180 bp PCR product seen on gels. A diagnostic assay capable of detecting aquatic birnaviruses directly in fish samples, would be of significant benefit for fish health management programs. But degradation of viral RNA by the nucleases in the tissue homogenates or visceral samples could invalidate the PCR method when the samples are sent by mail from the farm to the laboratory. Heparinized blood and leukocyte purification seems to accurately preserve the virus content of the cells; IPNV is detected by N-PCR or FCM at a level of accuracy and sensitivity comparable to those of virus

isolation in cell culture, but in less time. In this work

eight to twenty four hours were required to obtain results,

including the time for leukocyte purification and RNA

extraction, versus the 3 or 4 weeks necessary for

isolation and identification of IPNV by conventional

methods.

From the epizootiological perspective, it is important

that the causative agents of fish diseases are rapidly

differentiated, as determination of the origin of an

outbreak may help to prevent further introductions.

Leukocytes seem to be the target cells and FCM or PCR

the best choice to improve rapid detection of IPNV in fish

carriers.

Acknowledgments

This study was supported by grants from the Comision Interministerial de Ciencia y Tecnologla,

project AGF 98-0837 and Comunidad Autonoma de Madrid, project 079/23/98. We thank Mercedes Sanchez for her excellent technical assistance.

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