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. 1999 Jun;37(6):1858–1862. doi: 10.1128/jcm.37.6.1858-1862.1999

Evaluation of a Nested Reverse Transcription-PCR Assay Based on the Nucleoprotein Gene for Diagnosis of Spontaneous and Experimental Bovine Respiratory Syncytial Virus Infections

Jean-François Valarcher 1, Hervé Bourhy 2, Jacqueline Gelfi 3, François Schelcher 1,*
PMCID: PMC84970  PMID: 10325337

Abstract

The first nested reverse transcription (RT)-PCR based on the nucleoprotein gene (n RT-PCR-N) of the bovine respiratory syncytial virus (BRSV) has been developed and optimized for the detection of BRSV in bronchoalveolar lavage fluid cells of calves. This test is characterized by a low threshold of detection (0.17 PFU/ml), which is 506 times lower than that obtained by an enzyme immunosorbent assay (EIA) test (RSV TESTPACK ABBOTT). During an experimental infection of 17 immunocompetent calves less than 3 months old, BRSV RNA could be detected up to 13 days after the onset of symptoms whereas isolation in cell culture was possible only up to 5 days. Compiling results obtained by conventional techniques (serology, antigen detection, and culture isolation) for 132 field samples collected from calves with acute respiratory signs revealed that n RT-PCR-N showed the highest diagnostic sensitivity and very good specificity. This n RT-PCR-N with its long period of detection during BRSV infection thus provides a valuable tool for diagnostic and epidemiological purposes.

Bovine respiratory syncytial virus (BRSV), like human respiratory syncytial virus (HRSV), is a pneumovirus in the Paramyxoviridae family (7). The epidemiology of BRSV is very similar to that of HRSV (34). Spontaneous infection in young cattle is frequently associated with severe respiratory signs (10, 31, 37), whereas experimental infection generally results in milder disease with slight pathologic changes (4). Diagnosis of BRSV infection is based solely on antigen detection and serology (3). The method of choice in dead cattle consists of the detection of BRSV antigens in pulmonary samples by immunofluorescence (20) or immunoperoxidase (23) technique. BRSV antigen detection is less frequently undertaken in live cattle. Nasal secretion sampling (NS), which is slightly invasive and currently used for human infants, has sometimes been described in calves (3). Bronchoalveolar lavage (BAL) (21, 33) can be more sensitive, since the lung is usually extensively infected in naturally occurring cases (39). BRSV lability makes routine isolation in cell culture laborious and time-consuming (21). To our knowledge, comparison of sampling of the upper respiratory tract and lower respiratory tract has only rarely been conducted in infants (9) and no comparison is available in cattle. As previously demonstrated, in young calves under 3 months, maternally transmitted immunoglobulin (Ig) G1 does not prevent BRSV infection but hinders serological diagnosis (21, 33). However, the combined use of serology, antigen detection, and culture, which could potentially increase the proportion of positive diagnoses, is laborious and expensive.

Reverse transcription (RT)-PCR is potentially useful for improving the sensitivity of RSV detection. Several RT-PCRs for HRSV detection based on the fusion (F) gene (17, 28), the 1B gene (36), the polymerase (L) gene (13), and the nucleoprotein (N) gene (8, 15) have been developed and have been evaluated in studies involving numerous field specimens. The sensitivity results are controversial. Some authors consider RT-PCR less sensitive than expected when compared with conventional techniques of HRSV detection (8, 13, 28), whereas for others, the sensitivity was slightly (36) or more-strongly improved (15, 17). The RT-PCR tests reported for BRSV are directed against F (25, 26, 38) and glycoprotein (G) (38) genes. None based on the N gene from BRSV has been described even though this is one of the most conserved of the pneumovirus genes (19). Furthermore, these BRSV studies involved small numbers of strains, diseased calves, and infected herds (26, 38).

The aims of this work were therefore (i) to evaluate the threshold of detection of a nested RT-PCR developed based on the N gene (n RT-PCR-N), (ii) to determine the duration of presence of the BRSV in BAL fluid during an experimental infection, and (iii) to evaluate the sensitivity and specificity of the RT-PCR obtained during a large field study and compare them with those of currently used laboratory techniques (serology, antigen assays, and isolation in cell culture).

MATERIALS AND METHODS

Animals. (i) Spontaneous infections.

Between 1991 and 1998, 111 live calves and 21 dead calves (n = 132; average age, 58 days; minimum age, 6 days; and maximum age, 480 days) from 49 different herds were studied to compare sites of sampling and laboratory techniques used to diagnose BRSV infection. Calves were in cow-calf herds located in Belgium (9 herds) and in central and southwestern France (40 herds). Animals exhibited acute respiratory signs compatible with BRSV infection. Dead calves were necropsied within 36 h of death.

(ii) Experimental infection.

Seventeen male calves of Prim’Holstein breed, with a BRSV-seronegative status, were reared in isolation. When they were 2 to 3 months old, they were used for challenge experiments after their BRSV serological status had been confirmed to be negative. Eleven calves were inoculated with BRSV (BRSV A2 gelfi), and six calves were inoculated with bovine turbinate (BT) cells free of virus (controls), on 3 consecutive days (D0, D1, and D2). Each day, half of a 20-ml portion of inoculum containing no BRSV or 2.5 × 107 PFU of BRSV was instilled intranasally and the other half was injected endobronchially. BAL was performed at D2 on all the animals and then every 2 days for five calves from D4 to D18 and every 2 days for six calves from D5 to D19 and for some of the animals at D30 and D36 postinoculation (p.i.). A standardized clinical examination was carried out every day from D2 to D36, and the mean clinical score was calculated (12).

Viral strains.

A field BRSV strain (A2 gelfi) isolated in 1994 during a BRSV outbreak in France involving adults and calves was propagated in BT cells in minimum essential medium with Earle’s salt and glutamine (MEM) (Gibco BRL, Cergy Pontoise, France) supplemented with 3% fetal calf serum (FCS). The sixth passage was aliquoted and stored at −80°C. The titer was 1.25 × 106 PFU/ml. This stock provided the challenge inoculum and positive controls for PCR, isolation in cell culture, and antigen detection. The absence of common viral respiratory pathogens, including bovine viral diarrhea virus (BVDV), in the inoculum was checked.

BRSV isolates or strains W6 Toulouse, from France, FV160, 220/69, MRV533, 5761, and RB-94 from Belgium (kindly provided by G. Wellemans), Lelystad and V347, from The Netherlands (kindly provided by T. J. Kimman and and R. S. Schrijver), and 127, from the United Kingdom (kindly provided by T. J. Kimman), were used as strains of BRSV representative of the diverse strains found in Europe (29).

Infectious bovine rhinotracheitis virus (IBRV), parainfluenza virus 3 (PI3), and BVDV strains isolated at the Toulouse Veterinary School were used to check n RT-PCR-N specificity.

Clinical samples.

Clinical samples included serum, NS, BAL fluid cells, and lung samples. Two blood samples were collected at a 2- to 3-week interval, and the serum was stored at −20°C until analysis. Nasal cells and NS were collected with sterile cotton swabs, placed in storage at 4°C less than 2 h after collection, and stored until virological examination. Lung samples from necropsied calves were kept at 4°C for less than 1 h and then stored at −80°C. Virological examinations were simultaneously performed less than 1 month after sampling.

BAL was performed with a fiber-optic endoscope (GIF-Q; Olympus, Paris, France) via the mouth and under general anesthesia (ketamine hydrochloride, 3 mg/kg of body weight). The airways were successively washed three times with 50 ml of MEM, 1× nonessential amino acid (NEEA) (Gibco BRL), gentamicin (Gibco BRL) (200 mg/ml), enrofloxacin (BAYTRIL 5% injectable; Bayer, Puteaux, France) (0.2 μg/ml), and Fungizon (Gibco BRL) (2.5 μg/ml). The endoscopes were chemically decontaminated after each wash. The decontamination process was validated to check that no residual BRSV was recovered by n RT-PCR-N (data not shown). BAL fluid cells from field animals were stored in 95% FCS and 5% dimethyl sulfoxide (Sigma, Saint Quentin Fallavier, France) at −80°C until culture isolation or RT-PCR, both of which were performed within the first month following sampling. BAL fluid cells from experimentally infected animals were kept at 4°C and then seeded on BT cells less than 2 h after sampling for culture isolation. They were stored in TRIzol Reagent (Gibco BRL) at −80°C until analysis by n RT-PCR-N. In all cases, enzyme immunosorbent assays (EIA) and cell cytocentrifugation for indirect immunofluorescence (IIF) were performed just after sampling. Slides for IIF were stored at −20°C until immunolabelling.

Serology.

Paired serum samples were analyzed with a commercially available enzyme-linked immunosorbent assay (ELISA) kit (LSI/VRS; LSI, L’arbresle, France) according to the manufacturer’s recommendations. The increase in antibodies was considered significant when the corrected optical density at 450 nm (cOD) of the second serum sample was ≥0.2 and when the ratio of the cOD of the second serum sample to the cOD of the first serum sample (collected 2 to 3 weeks before) was ≥2.

BRSV isolation on BT cells.

For BRSV culture isolation, BT cells were cultivated at 37°C in 5% CO2 and in culture medium (CM) (MEM [Gibco BRL] containing NEAA [Gibco BRL], penicillin [Gibco BRL] [100 IU/ml], dihydrostreptomycin [Gibco BRL] [100 μg/ml], Fungizon [Gibco BRL] [2.5 μg/ml], and 3% FCS). The cells contained in 10 ml of BAL fluid or stored in FCS and dimethyl sulfoxide were centrifuged (1,000 × g for 10 min at 4°C), frozen in 1 ml of CM, thawed, and then seeded on BT cells. For virus isolation from lung, 1 cm3 from the densified part (approximately 4 g) was crushed in a mortar and suspended in 10 ml of the medium used for BT cell culture. After centrifugation (700 × g for 10 min at 4°C), 1 ml of the supernatant was seeded on BT cells. After 2 h of adsorption, the inoculum was removed and the BT cells were washed once with 5 ml of CM and incubated with 5 ml of fresh CM. To eliminate bacterial contamination, the BT cells were washed once again 24 h after inoculation. If bacterial contamination was observed the BT cells were washed twice within the next 12 h. Three subculture passages were made in the absence of any characteristic cytopathic effect. BRSV was identified by an IIF test by using the monoclonal antibody 18 B2 directed against the F protein of HRSV (Argen Biosoft, Varilhes, France), which cross-reacts with BRSV (23). The secondary antibody was an anti-mouse rabbit polyclonal antibody-labelled fluorescein isothiocyanate (Sanofi Pasteur, Aulnay sous bois, France).

Antigen detection.

The detection of BRSV antigen was carried out by IIF (with the same antibodies as described for identification of BRSV on BT cells) or by a commercial EIA, EIA RSV TESTPACK ABBOTT (EIA-TP) (Abbott, Rungis, France). IIF was performed on cytospin preparations of BAL fluid cells or on cryosections of lung. For cytospin, BAL fluid cells were filtrated through gauze, adjusted to a total number of 5 × 104, and cytocentrifuged (450 rpm for 8 min) by using a Cytospin 3 (Shandon, Pittsburgh, Pa.). EIA-TP was performed according to the manufacturer’s recommendations on BAL fluid cells contained in 10 ml of BAL fluid, on NS, or on clarified homogenate of lung used for culture isolation.

RNA extraction.

The procedure for RNA extraction was adapted from a method previously described (6). RNA extractions were performed on BAL fluid cells contained in 10 ml of BAL fluid and on approximately 1 g of crushed lung sample. Each sample was frozen at −80°C in 1 ml of a solution of phenol and guanidine isothiocyanate (TRIzol Reagent; Gibco BRL). It was then thawed and incubated at room temperature for 5 min, and 250 μl of chloroform was added to each sample. The tubes were centrifuged (12,000 × g for 15 min at 4°C) after vigorous shaking for 2 min and then allowed to stand at room temperature for 10 min. The supernatant was collected, and 1 ml of a more-concentrated solution of phenol and guanidine isothiocyanate (Trizol LS Reagent; Gibco BRL) was added to each tube. After purification by chloroform (vol/vol) and DNA decontamination by isopropanol alcohol (1/10 volume), the RNA was precipitated in isopropanol (vol/vol) with 0.3 M sodium acetate (pH 5.2) (Sigma) and 2 μl of Pellet Paint Co-Precipitant (Novagen, Abingdon, England) overnight at 4°C. The sample was then centrifuged (12,000 × g for 10 min at 4°C), the supernatant was removed, and the pellet was washed in 1 ml of 70% ethanol. The sample was again centrifuged (7,500 × g for 5 min at 4°C), the supernatant was removed, the pellet was dried for 10 min, and the RNA was resuspended in 36 μl of diethylpyrocarbonate-treated (DEPC) water.

RT-PCR.

Primers used for RT-PCR-N were based on a multiple alignment, performed by using the ClustalW 1.60 program (18), of available sequences of N genes of RSVs. These sequences were from the following strains: three BRSV strains, RB-94 isolated in Belgium in 1970, A51908 isolated in Maryland in 1975, and 391.2 isolated in North Carolina in 1985 (GenBank accession no. L27840, M35076, and S40504, respectively); one ovine RSV (GenBank accession no. U07233); and two HRSV strains representing the HRSV subtypes A and B, strain A2 and strain 18537 (GenBank accession no. M11486 and D00390, respectively). PRIMER version 0.5 (22) was used for primer selection to ensure an optimal primer length of 20 nucleotides, to be compatible with the annealing temperature, and to have no more than eight bases of self-complementarity or no more than four bases of self-complementarity in the 3′ terminal matching region. Primers were selected according to the consensus sequence of BRSV strain in conserved regions with HRSV strains and ovine RSV.

For RT, 1 μl (2 pmol) of primer N2.1 (5′-ATGGCTCTCAGCAAGGTCA-3′; positions 1 to 19 on the N coding region of the BRSV genome [30]) mixed with 9 μl of DEPC-treated water containing RNA was incubated at 68°C for 10 min and then at 58°C for 10 min and finally chilled on ice. Each tube received 4 μl of a solution containing each nucleotide triphosphate (10 mmol), 1 μl of RNasin (40 U) (Promega, Charbonnières, France), 2 μl of dithiothreitol (0.1 M) (Gibco BRL), 4 μl of SuperScript TM II buffer (250 mM Tris HCl, 375 mM KCl, 15 mM MgCl2) (Gibco BRL), and 1 μl of SuperScript TM II (200 U) (Gibco BRL) and was incubated at 42°C for 50 min. The reverse transcriptase was inactivated by heating at 70°C for 15 min. The RNA-cDNA hybrids were diluted 10 times in DEPC-treated water. Ten microliters of diluted cDNA was mixed in a final volume of 100 μl with 5 μl of primer N2.1 (50 pmol) and 5 μl of primer N2.2 (50 pmol) (5′-TCTTGGTTTCTTGGTGTACCTC-3′; positions 1034 to 1013 on the N coding region of the BRSV genome [30]) and used for the first round of PCR, which was carried out in a solution containing 200 μM of each nucleotide triphosphate, 10 mM Tris HCl (pH 8.3), 50 mM KCl, 1 mM MgCl2, and 2.5 IU of AmpliTaq Gold polymerase (Perkin Elmer Cetus, Roissy-Charles de Gaulle, France). The first round of PCR, which amplified 1,034 nucleotides (nt), was performed in a GeneAmp PCR System 480 (Perkin Elmer Cetus) by using the following program: 94°C for 12 min followed by 35 cycles of denaturation at 94°C for 60 s, annealing at 58°C for 60 s, and elongation at 72°C for 90 s and ending with a final elongation for 10 min. Ten microliters of the PCR products diluted 10 times was used to perform the second round of PCR with the same mix but containing the internal primers N2.3 (5′-CATCTCAATAAGTTGTGTGG-3′; positions 127 to 146 of the N coding region of the BRSV genome [30]) and N2.4 (5′-TCTACAACCTGTTCCATTTC-3′; positions 857 to 838 on the N coding region of the BRSV genome [30]). The second round of PCR amplified 731 nt and used the following program: 94°C for 12 min followed by 35 cycles of denaturation at 94°C for 45 s, annealing at 49°C for 60 s, and elongation at 72°C for 60 s and ending with a final elongation for 10 min.

The PCR products were detected by electrophoresis on a 2% agarose gel containing ethidium bromide (0.1 μg/ml).

RESULTS

Threshold of detection of n RT-PCR-N.

In order to improve the detection in BAL samples, an n RT-PCR was developed to amplify a region of the N gene. Different preliminary tests concerning the number of PCR cycles (it was found that 35 cycles produced better results than 25) and hybridization temperatures (it was found that for the first step 58°C produced better results than 56 and 60°C and for the second step 49°C produced better results than 50 and 52°C) were performed to optimize the protocol. When optimized, the threshold of detection for the n RT-PCR-N was determined by four independent serial 10-fold dilutions of the suspension of virus A2 gelfi. It was 506 times lower (mean = 0.17 PFU, standard deviation [SD] = 0.16 PFU) than that for EIA-TP (mean = 88.12 PFU, SD = 16.88 PFU).

Duration of detection of BRSV in BAL fluid during an experimental infection.

Eleven calves were inoculated with BRSV, and six calves were mock infected. All the animals in the inoculated group developed clinical signs. The mean clinical score from D5 to D13 p.i. was significantly higher (P < 0.001) in the BRSV-inoculated group. The first clinical signs were observed on D4 and D5 p.i., and individual scores increased to a maximum at D7 or D8 p.i. and decreased slowly thereafter. One hundred and seven BAL fluid samples were sequentially obtained from BRSV-infected calves from D2 to D36 p.i. The results obtained by n RT-PCR-N, isolation on cell culture, and IIF were compared (Table 1). None of the six control calves was positive during the trial. All the inoculated calves were identified as positive by each of the three techniques during the acute phase of the disease (D4 to D7 p.i.). At D8 and D9 p.i., 4 (36.4%) of 11 samples and 3 (50.0%) of 6 samples were found to be positive by isolation on cell culture and IIF, respectively, whereas all 11 samples (100%) tested by n RT-PCR-N were positive. After the 9th day p.i., all the results for IIF and isolation on cell culture were negative. BRSV was detected until D17 p.i. in 2 of 11 animals (17%) by RT-PCR. BRSV was not detected at D30 and D36 p.i. The average duration of detection of BRSV in BAL fluid by n RT-PCR was 12.7 days (n = 11). This was significantly longer than the duration of detection by IIF (8.0 days, n = 6) or by isolation (7.3 days, n = 11) (P < 0.01, χ2 test).

TABLE 1.

Comparison of positive results obtained by n RT-PCR performed by using the N gene, isolation in cell culture, and IIF of BAL cells collected at various times after experimental infection of calves with BRSV

Method No. of positive results/no. of specimens tested on indicated day p.i.
−2 4 or 5 6 or 7 8 or 9 10 or 11 12 or 13 14 or 15 16 or 17 18 or 19 30 36
n RT-PCR 0/11 11/11 11/11 11/11 11/11 7/11 2/11 2/11 0/11 0/5 0/3
Isolation on cell culture 0/11 11/11 11/11 4/11 0/11 0/11 0/11 0/11 0/11 0/5 0/3
IIF 0/6 6/6 6/6 3/6 0/6 0/6 0/6 0/6 0/6 NDa ND
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a

ND, not done. 

Comparison of n RT-PCR-N, serology, EIA-TP, IIF, and isolation on cell culture for field specimens.

During the acute phase of a respiratory syndrome characterized by signs or lesions compatible with BRSV infection, n RT-PCR-N (with BAL fluid cells and lung), EIA-TP (with NS, BAL fluid cells, and lung), IIF (with BAL fluid cells), and serology were performed by using field specimens obtained from a total of 132 calves. To ensure clarity of the results, EIA-TP, IIF, isolation on cell culture, and n RT-PCR-N were successively chosen as the reference technique to which each of the other techniques was compared. The numbers of samples used for each of these comparisons were not the same. For each of these comparisons the prevalence calculated on the basis of the reference technique and the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of each of the confirmatory techniques were determined (Table 2).

TABLE 2.

Comparison of BRSV diagnostic assays on field samples from calves with clinical signs or lung lesions compatible with BRSV infection

Laboratory techniques
Type of samplea No. of animals No. of samples with indicated result for assay 1/assay 2
Prevalence according to assay 1 (%) Sensitivityc (%) Specificityc (%) PPVc (%) NPVc (%)
Assay 1 Assay 2 +/+ +/− −/+ −/−
EIA-TP EIA-TP BAL fluid cells/NS 83 15 6 0 62 25.3 71.4 100.0 100.0 91.2
EIA-TP Serologyb BAL fluid cells/serum 83 8 13 2 60 25.3 38.1 96.8 80.0 95.2
IIF BAL fluid cells 83 19 2 1 61 25.3 90.5 98.4 95.0 96.8
IIF Lung 16 14 0 0 2 87.5 100.0 100.0 100.0 100.0
IIF Serologyb BAL fluid cells/serum 83 7 13 3 60 24.1 35.0 95.2 70.0 95.2
Culture Lung 13 1 10 0 2 84.6 9.1 100.0 100.0 16.7
Culture EIA-TP BAL fluid cells 28 19 1 0 8 71.4 95.0 100.0 100.0 88.9
n RT-PCR EIA-TP BAL fluid cells 28 19 4 0 5 82.1 82.6 100.0 100.0 55.0
EIA-TP Lung 21 17 2 0 2 90.5 89.5 100.0 100.0 50.0
Culture BAL fluid cells 28 20 3 0 5 82.1 87.0 100.0 100.0 62.5
Culture Lung 13 1 10 0 2 84.6 9.1 100.0 100.0 16.7
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a

When two different samples were used, they are indicated as follows: sample type of assay 1/sample type of assay 2. 

b

Serology was considered positive when a significative increase of antibodies for the second of two blood samples collected at a 2- to 3-week interval was observed. 

c

Obtained for assay 2 when assay 1 is taken as the reference. 

EIA-TP performed with NS were less-frequently positive (but not significantly) than those performed with BAL fluid cells, i.e., 15 of 83 samples (18.1%) and 21 of 83 samples (25.3%), respectively, were positive (Table 2). A significant increase in blood BRSV antibodies was observed in only 10 of 83 calves (12%). When EIA-TP with BAL fluid cells was chosen as the “gold standard,” serology had a very low sensitivity of 0.38 and a specificity of 0.97 (n = 83). Thirteen calves that were negative by serology assay because they already had a high level of specific antibodies at the time of collection of the first serum sample were shown to be positive by EIA-TP or by IIF on BAL fluid cells. The sensitivity of detection of antigen on BAL fluid cells (IIF or EIA) was significantly higher (P < 0.05, χ2 test) than that of serology (Table 2). Moreover, three calves were positive by serology assay and negative by antigen detection (Table 2). IIF and EIA-TP with BAL fluid cells gave similar results except for three calves (Table 2). In contrast to the calf positive only by IIF, the only two calves that were positive by EIA-TP could be considered true positives according to their serological results or by the detection of BRSV in other calves from the same herd.

For 28 field BAL fluid samples, the comparison of cell culture and EIA-TP gave similar results (Table 2). It might seem that the highest sensitivity was obtained by isolation on cell culture followed by EIA-TP but these results were not significantly different (χ2 test). The results obtained with necropsy specimens of lungs were slightly different from those obtained during the life of the calves. BRSV detection, with 16 lungs, by IIF and by EIA-TP gave identical results (Table 2). However, isolation on cell culture had a significantly lower sensitivity than IIF and EIA-TP (P < 0.01, χ2 test) and a low NPV (Table 2).

The nested RT-PCR was then compared to EIA-TP (28 BAL fluid cell specimens and 21 lungs) and to isolation on cell culture (28 BAL fluid cell specimens and 13 lungs) for 49 naturally occurring cases of respiratory disease in young cattle (Table 2). For BAL fluid cells, results were in concordance except for four samples which were positive by n RT-PCR-N and negative by EIA-TP. Three of them were also negative by isolation on cell culture. Nineteen of 21 lungs were found to be positive by n RT-PCR-N; among them 17 were simultaneously identified as positive by EIA-TP.

Isolation on cell culture was also attempted on 13 of the 21 lungs. Eleven of these were simultaneously positive by IIF, EIA-TP, and n RT-PCR-N. However, BRSV was isolated from only one of these lungs. Compared to n RT-PCR-N, the sensitivity of isolation on cell culture was 0.87 for BAL fluid cells and 0.09 for lung.

The spectrum of detection of the n RT-PCR-N was also verified by using nine other field isolates (W6, FV 160, 220/69, MRV533, 5761, RB-94, Lelystadt, V347, and 127) originating from three European countries. All of them were correctly amplified. The specificity of this n RT-PCR assay was also tested by using different pathogens (IBRV, PI3, and BVDV) not related to BRSV and associated with common respiratory disorders. The results were negative.

DISCUSSION

Differences in prevalence (ranging between 25.3 and 90.5%) were observed in our field study depending on the series of calves studied. These differences can be explained by numerous factors, such as choice of herds and animal inclusion or diagnostic tools.

Nasal swabs and, even more often, nasopharyngeal aspirates rather than BAL fluid are commonly used to detect HRSV in infants (1). However, in BRSV infections (this study) as in HRSV infections (9), the sensitivity of EIA-TP on BAL fluid seems higher. Several hypotheses can be put forward. RSV is currently recovered from alveolar macrophages (24). Nasal swabs may sometimes collect an insufficient quantity of infected mucosal cells. However, nasal sampling is less invasive and easier to perform than BAL.

Results obtained by EIA-TP, IIF, and culture isolation on BAL fluid cells were similar. Furthermore, the sensitivities of EIA-TP and IIF were similar and significantly higher than that obtained for culture isolation by using lung specimens. These data could be explained by the lability of the virus several hours after death. Antigen capture ELISAs, such as EIA-TP, are frequently used in the diagnosis of HRSV infection in infants. Many of the antibodies developed against HRSV cross-react with BRSV (27). Results are easier and faster to obtain by EIA-TP than by IIF (assay times, 30 min and 4 h 30 min, respectively).

The sensitivity of antigen detection (IIF or EIA-TP) appeared to be significantly higher than that obtained by serology, thus confirming previous results (5, 21, 33). This low sensitivity of serology can be explained by the interference between maternally acquired antibodies and postinfection immune response (21). Specific IgM ELISA is potentially useful for overcoming this problem (40). However, in some calves no antigens were detected although a seroconversion was observed. Furthermore, respiratory signs could in some cases persist without BRSV detection. This suggests that antigen detection gives positive results only during a short time, which coincides with the acute phase of respiratory disease (4). More-sensitive diagnostic tools are thus needed to monitor BRSV infection.

The high conservation of the N gene in HRSV (19) and BRSV (2, 30) enabled us to define efficient and conserved primers. We therefore developed and optimized a nested RT-PCR whose target was the N gene to ensure high sensitivity and specificity. The ability of this n RT-PCR-N to detect a very small amount of virus was demonstrated by the very low threshold of detection (0.17 PFU/ml). The duration of the detection of BRSV in BAL fluid cells was investigated during an experimental infection of calves, which developed moderate to severe clinical signs. The duration of shedding obtained by n RT-PCR-N (13 days) was approximately more than twice that by isolation on cell culture or by IIF (5 days). Other authors reported that the detection of BRSV by isolation in cell culture (4, 32, 35) or by n RT-PCR targeting the F gene (11, 38) for more than 6 days was not possible. According to our data, the period of shedding of BRSV in calves is thus similar to that of HRSV in infants (8). Whether BRSV can be detected for as long as HRSV in immunocompromised infants (14, 16) is still open to debate.

The sensitivities and specificities of different laboratory techniques were also analyzed by using a series of clinically and epidemiologically identified field specimens. The sensitivity of n RT-PCR-N was higher than those of all the other techniques tested. We did not find any specimen identified as positive by conventional assay and negative by RT-PCR-N, in contrast to other reports for HRSV (13, 15, 17). These results could be linked to the elimination of potential inhibitory factors or to the fitness of the primers to the RNA template. On the other hand and despite potential RNA degradation, identification of BRSV RNA on necropsied lung samples was possible until 36 h after death. The specificity of n RT-PCR-N was checked. No amplification could be observed when n RT-PCR-N was performed by using control BAL fluid cells in the experimental model or the different common respiratory pathogens. Moreover, the sequences of portions of amplified N, G, and F genes were determined for each of 13 instances of discordance between results of n RT-PCR-N and isolation from BAL fluid cells or lungs. Pairwise comparisons indicated that these sequences differed by at least 1 nucleotide (not shown). This strongly supports the idea that a different BRSV isolate is present in each of the specimens.

In conclusion, the n RT-PCR presented in this study seems to be more sensitive than and at least as specific as IIF, EIA, or isolation in cell culture. It will allow (i) a broadening of the detection period for clinical specimens and (ii) study of the molecular epidemiology of field BRSV isolates.

ACKNOWLEDGMENTS

We are grateful to S. Bonhoure, F. Lasserre, and M. Moulignié for their technical assistance. We thank S. Bertagnoli for helpful discussion and D. Desmecht for lung specimens.

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