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K. KOVAØÈÍK
ABSTRACT: We developed an indirect enzyme-linked immunosorbent assay (ELISA) for detection of serum antibodies
to bovine respiratory syncytial virus. For evaluation of the newly developed ELISA, field sera collected from 549 head of
cattle in the Czech Republic were tested in parallel by a serum neutralization test. The tests showed 98.36% agreement.
The specificity and sensitivity of the ELISA relative to serum neutralization test was 97.00% (226/233) and 99.37%
(314/316), respectively. Tissue culture-grown viral antigen was used in the tests. The corrected optical density (COD) of
each sample tested at dilution 1/100 was expressed as a percentage of the COD of a positive reference serum included on
each plate, this value was the sample/positive (S/P) ratio. We determined the relationship between the S/P ratio (%) ob-
tained at a dilution 1/100 and the end point titer calculated by serum neutralization test (r = 0.9743). The ELISA test was
evaluated by testing acute and convalescent (3 wk later) serum pairs from 9 head of cattle with confirmed BRSV infec-
tion for demonstration of seroconversion. The ELISA test demonstrated a clear increase of the S/P ratio (%) between
acute and convalescent serum pairs (on average 42.2 ± 13.1).
Keywords: bovine respiratory syncytial virus (BRSV); respiratory tract pathogen; cattle; ELISA
A list of abbreviations: BRSV bovine respiratory syncytial virus, SN test serum neutralization test, HIT hae-
magglutination inhibition test, FAT fluorescent antibody test, CPE cytopathic effect, OD optical density, PBS-T
phosphate buffered saline with Tween 20, MEM minimal essential medium, RT-PCR reverse transcription-polymerase
chain reaction.
INTRODUCTION
method. The most frequent diagnostic methods are sero-
A complex of mass diseases of the respiratory system logical methods serum neutralization test (Baker et al.,
referred to as respiratory syndrome is the cause of consid- 1986), complement fixation test, indirect fluorescent anti-
erable economic losses in calf and young cattle rearing. body test and ELISA test (Gillette, 1983; Elvander et al.,
The Bovine Respiratory Syncytial Virus (BRSV) is one 1995). BRSV was isolated in a cattle herd in the Czech Re-
of the most important and, in the Czech Republic, some- public in 1996 from an acute outbreak of respiratory dis-
what underestimated causes of the respiratory syndrome ease (Kovaøèík, 1999). Between 1998, and 1999 the BRSV
in calves and young cattle. infection was also serologically diagnosed (SN test) in
BRSV was first isolated in 1967 in Switzerland (Pac- other herds affected by serious respiratory disease. Despite
caud and Jaquier, 1970) from calves with a respiratory efforts to perfect micromethods, SN test remains a time-
tract disease. In the Czech Republic, the virus was success- consuming technique with difficult result evaluation and
fully isolated in 1976 from an acute outbreak of respira- is therefore used rather in research studies than in rou-
tory disease in feedlot cattle (Pospíil et al., 1978). Both tine diagnostics. This is why the indirect ELISA test was
the isolation of the virus and serological studies suggest developed for the detection of antibodies to BRSV in
that BRSV causes outbreaks of acute respiratory diseases blood serum. This test simplifies the diagnostics of BRSV
worldwide (Edington and Jacobs, 1970; Wellemans et al., infections and can be used as a routine diagnostic
1970; Jacobs and Edington, 1971; Köves and Bartha, method.
1975). This article describes the development and use of the
BRSV is unusually sensitive to the outer environment ELISA test for demonstrating BRSV anibodies and com-
and its isolation from clinical specimens is very excep- pares the specificity and sensitivity of the newly devel-
tional and therefore is not recommended as a routine oped ELISA test with a SN test.
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Original Paper Vet. Med. Czech, 46, 2001 (2): 2934
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Vet. Med. Czech, 46, 2001 (2): 2934 Original Paper
suspension containing approximately 100 TKID50 /0.1 ml and soaked for 1 hour at 4°C while being shaken thor-
of BRSV. After incubating for 1.5 hours at room tempera- oughly from time to time. The PBS including the cellular
ture, a suspension of secondary fetal calf kidney cells in detritus was then centrifuged at 14 000 rpm for 10 seconds
MEM Eagle with 30% of precolostral bovine serum was in an Eppendorf centrifuge. The pellet was re-suspended
added in the volume of 50 ml (5 × 105 cells/ml). The plates in 50 µl of PBS (pH 7.6) and transferred onto a slide.
were incubated at 37°C in 5% CO2 humidified atmosphere After air drying, the cellular detritus was fixed in cooled
for 5 to 6 days. Serum titres were established as recipro- acetone for 10 minutes. After washing in PBS for 10 min-
cal values of the highest serum dilution which com- utes, conjugate was added in a 1 : 20 dilution (manu-
pletely inhibit CPE in at least one out of two wells. facturers recommendation). Incubation took place in
a moist chamber at 37°C for 30 minutes. Following the
incubation, the slide was rinsed in PBS three times for
Detection of antibodies to other bovine viruses 5 minutes. A PBS/glycerol mixture was transferred onto
plates with the cellular detritus and it was then covered
Commercial ELISA tests have been used to detect an- with a cover-glass.
tibodies to the infectious bovine rhinotracheitis (IBR) Results were read with the help of a fluorescent micro-
and bovine virus diarrhoea virus (BVDV) according to scope.
the manufacturers instructions (Test-Line Ltd., Clinical
Diagnostics, Brno, Czech Republic). A standard haemag-
glutination-inhibition test (HIT) was used to detect anti- Isolation and identification of the virus
bodies to the parainfluenza virus 3 (PI-3).
The isolation and identification of the virus were car-
ried out according to a previously described method
Haemagglutination-inhibition test (Kovaøèík, 1999).
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Original Paper Vet. Med. Czech, 46, 2001 (2): 2934
Nine serum samples were examined from animals the antibodies to the BVD virus and all nine samples
which were in the acute phase of the disease and nine se- were positive for the antibodies to the PI-3 and IBR vi-
rum samples from animals in the convalescent phase (after ruses. The convalescent serum samples had the same re-
3 weeks) of the BRSV infection. Only 1 serum sample sults as the sera in the acute phase of the disease and no
which had been collected in the acute phase of the disease active seroconversion (an increase of the specific antibody
was positive. All serum samples which had been collected level) against the BVD, IBR and PI-3 viruses was observed.
in the convalescent phase of the disease were positive
(Table 2). The increase of S/P ratios (%) in the convales-
cent sera amounted to 42.2 ± 13.1 (average ± standard Establishing a dependency between a SN titre and S/P ra-
deviation). tio set by the ELISA test
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Vet. Med. Czech, 46, 2001 (2): 2934 Original Paper
80
S/P (%)
60
40
r = 0.9743
n = 43
20
0
-1 0 1 2 3 4 5 6 7
This correlation coefficient is of high statistical signifi- the possible causes of such variance could be an ana-
cance (P < 0.01). The graphic demonstration of the regres- lytical error of the method. The other cause could be di-
sive straight line and the calculation of the correlation versity in the concentration of neutralizing and total
coefficient were both achieved using the Microsoft Ex- BRSV-reactive antibodies in naturally infected cattle and
cel 97 programme. following the administration of inactivated vaccines of
various origin (Ellis et al., 1995). The ELISA test detects
total BRSV-reactive antibodies (including those which
Analytical error of the ELISA method are incapable of neutralization) whereas SN test only de-
tects neutralizing antibodies. Three questionable samples
The analytical error of the intra-assay ELISA test ex- originated from one herd of adult animals between the
pressed in percent according to the formula: 100 × [ana- ages of 18 to 36 months where an inactivated BRSV vac-
lytical error of method (S)/mean value of absorbance cine was used.
(alternatively of S/P ratio)] comes to 8.1%. The analyti- In order to be able to use a serological method in prac-
cal error of the inter-assay ELISA method expressed in tice, it is essential to detect changes in antibody titers
percent according to the same formula is 19.7% for the following an infection caused by BRSV. Some studies
absorbance and 14.3% for the S/P ratio. state that the ELISA test failed in demonstrating sero-
The analytical error of the method was established from conversion and SN test proved to be the most effective
119 duplicates of serum samples. (Gillette, 1983). The cause of the ELISA test failure in
demonstrating seroconversion following an experimental
infection may be the use of class-specific (anti-IgG) con-
DISCUSSION jugate which is incapable of detecting class IgM antibodies.
After experimentally infecting non-colostral calves,
Serological methods belong among the most frequently IgM-class antibodies were detected 8 to 10 days post in-
used diagnostic methods for infections caused by BRSV fection (PI), while IgG1-class antibodies were detected
because a direct virus isolation on cellular cultures is dif- 13 to 17 days PI (Kimman et al., 1987). Similar results
ficult and time-consuming. The ELISA test is generally were obtained in another experiment where IgG1-class
considered to be a specific and sensitive method for de- antibodies were detected 10 to 16 days PI. IgM-class anti-
tecting serum antibodies to BRSV in comparison with bodies were detected 10 days PI, they reached their peak
the precise and sensitive serum-neutralizing test. on the 16th day PI and after that their level fell sharply
Our results obtained by comparing the ELISA test until the 38th day after infetion when they were unde-
and SN test demonstrated 99.37% sensitivity, where tectable (Florent and Wiseman, 1990). In both experiments
2 samples which were positive in SN test were negative in IgM and IgG1 class antibodies were detected at approxi-
the ELISA test (Table 1). The specificity came to 97.00% mately the same time. Titers of IgG1 antibodies were very
as 7 samples were negative in SN test but positive in the low for one week, while titers of IgM antibodies quickly
ELISA test. Absorbance values of these serum samples increased. For this reason the results obtained through
were low (0.2090.339). There are two possible explana- the ELISA test using class-specific conjugates (anti-IgG)
tions for the variance in the results of the 9 samples. One of in combination with a low number of samples need not
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Original Paper Vet. Med. Czech, 46, 2001 (2): 2934
lead to the unambiguous establishment of Florent G., Wiseman A. (1990): An IgM specific ELISA for
seroconversion. In the ELISA test we used rabbit conju- the serodiagnostics of viral bovine infections. Comp. Immunol.
gate against bovine immunoglobulins (both IgG and Microbiol. Infect. Dis., 13, 203208.
IgM), by which means we gained relatively unambiguous Gillette K.G. (1983): Enzyme-linked immunosorbent assay
results in demonstrating seroconversion following natural used to monitor serum antibodies to bovine respiratory syn-
infection (Table 2). The comparison of paired samples of cytial virus: comparison with complement fixation and neu-
blood sera may be carried out within the framework of tralization tests. Am. J. Vet. Res., 44, 22512255.
one test (intra-assay) using the corrected optical density Henkel J.H., Aberle S.W., Kundi M., Popow-Kraupp T. (1997):
of the samples. Within the framework of two tests sepa- Improved detection of respiratory syncytial virus in nasal
rated in time (interassay) it is more appropriate to com- aspirates by seminested RT-PCR. J. Med. Virol., 53, 366371.
pare the use of S/P ratios, which has the advantage of a Jacobs J.W., Edington N. (1971): Isolation of respiratory syn-
lower analytical error of the method. These indirect cytial virus from cattle in Britain. Vet. Rec., 88, 694.
methods of diagnosing infection may be supplemented Kimman T.G., Westebrink F., Schreuder B.E.C., Straver P.J.
by direct methods. Given that the isolation of BRSV is (1987): Local and systematic antibody response to bovine
difficult and time-consuming, it is not recommended as a respiratory syncytial virus infection and reinfection in calves
routine method. For this reason it is often replaced by with and without maternal antibodies. J. Clin. Microbiol.,
methods of direct immunofluorescence with monospe- 25, 10971106.
cific serum in nasopharyngeal samples (McNulty et al., Kovaøèík K. (1999): Isolation of bovine respiratory syncytial
1983) or in sections of lung fragments, especially from virus during an outbreak of acute respiratory disease in calves.
the apical and cardial lobes (Thomas and Stott, 1981). Vet. Med. Czech, 44, 121127.
Recently new diagnostic methods for detecting BRSV Köves B., Bartha A. (1975): Isolation of respiratory syncytial
have also been developed, for example RT-PCR (Vilcek et (RS) virus from cattle diseased with respiratory symptoms.
al., 1994; Henkel et al., 1997). Acta Vet. Acad. Sci. Hung., 25, 357362.
The indirect ELISA test is a a fast and reliable method McNulty M.S., Bryson D.G., Allan G.M. ( 1983): Experimental
for detecting BRSV antibodies in blood serum, permitting respiratory syncytial virus pneumonia in young calves: micro-
its routine use in the diagnosis of infections caused by biologic and immunofluorescent findings. Am. J. Vet. Res.,
BRSV. 44, 16561659.
Paccaud M.F., Jaquier C. (1970): A respiratory syncytial virus
of bovine origin. Arch. Ges. Virus Forsch., 30, 327342.
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Received: 010226
Accepted after corrections: 010308
Corresponding Author:
MVDr. Kamil Kovarèík, Veterinary Research Institute, Hudcova 70, 621 32 Brno, Czech Republic
Tel. +420 5 41 32 12 41, fax +420 5 41 21 12 28, e-mail: kovarcik@vri.cz
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