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Veterinary Quarterly Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tveq20
Diagnosis of swine influenza with an immunofluorescence technique using monoclonal antibodies a
a
a
M. Onno , A. Jestin , P. Vannier & C. Kaiser
b
a
Ministère de l'Agriculture , CNEVA , Station de Pathologie Porcine, B.P. n° 9, Ploufragan, 22440, France b
CNEVA, Laboratoire Central de Recherches Vétérinaires , Ministère de l'Agriculture , B.P. n° 67, Maisons, Alfort Cedex, 94703, France Published online: 01 Nov 2011.
To cite this article: M. Onno , A. Jestin , P. Vannier & C. Kaiser (1990) Diagnosis of swine influenza with an immunofluorescence technique using monoclonal antibodies, Veterinary Quarterly, 12:4, 251-254, DOI: 10.1080/01652176.1990.9694274 To link to this article: http://dx.doi.org/10.1080/01652176.1990.9694274
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sublicensing, systematic supply, or distribution in any form to anyone is expressly
Downloaded by [University of Kent] at 08:59 08 December 2014
forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Diagnosis of swine influenza with an immunofluorescence technique using monoclonal antibodies M. Onnol, A. Jestinl, P. Vannierl, C. Kaiser2
Downloaded by [University of Kent] at 08:59 08 December 2014
SUMMARY Direct diagnosis of swine influenza infection by an indirect immunofluorescence technique using anti-nucleoproteine monoclonal antibody was compared with virus isolation. Sw/4115/85. Clinical Five 8-week-old pigs were inoculated with 2 x 107 EID50 of strain A H1N1Sw/ signs developed in only three pigs. Antigen was detected in nasal epithelial cells obtained from
all animals the first day after inoculation; the antigen was detected in one pig 6 days after the infection. Fluorescence was present in the nucleus, nucleolus and cytoplasm of infected cells. The indirect immunofluorescence test was specific and as sensitive as virus isolation in embryonated eggs, allowing a rapid diagnosis that could be achieved within hours.
INTRODUCTION
The laboratory identification of swine influenza by virus isolation or serology is still rather time-consuming and most often allows only a retrospective diagnosis. We thought that the rapid detection tests and the reagents used for the diagnosis of influenza in humans would be suitable for the rapid diagnosis of influenza in swine.
The immunofluorescence (IF) technique is commonly used to make a rapid diagnosis of influenza infection by detecting specific viral antigens in the desquamated epithelial cells (2, 3, 4, 7). Blaskovic made an attempt to obtain an early diagnosis of influenza in experimentally infected pigs by using an indirect immunofluorescent test and polyclonal antibodies (1). Diagnosis of influenza virus infections in swine by the fluorescent antibody technique on lung tissue of dead animals was reported in 1987 by Pensaert (5). The production of adequate monoclonal antibodies would considerably improve
the IF test. Such assays have already been performed in humans (7) and the sensitivity of the indirect IF test using anti-nucleoproteine (NP) monoclonal antibodies has been found to be higher than that of virus isolation. We selected an indirect IF test with anti-NP monoclonal antibodies in order to detect both HINI and H3N2 infections in nasal swabs from experimentally infected pigs.
1
MATERIALS AND METHODS
Virus and experimental infection
The swine influenza A HIN, strain no 153 (ref. OMS Sw/4115/85) was inoculated intratracheally (107 EID50) in five 8-week-old-specific-pathogen-free Large-White pigs. Five control animals were inoculated with MEM medium. Nasal swabs were collected daily for seven days post-inoculation. An aliquot of the samples was inoculated in the amniotic cavity of embryonated eggs. I
2
Ministere de l'Agriculture, CNEVA, Station de Pathologie Porcine, B.P. n° 9, 22440 Ploufragan,
France. Ministère de l'Agriculture, CNEVA, Laboratoire Central de Recherches Vétérinaires, B.P. n° 67, 94703 Maisons, Alfort Cedex, France.
THE VETERINARY QUARTERLY, VOL. 12,
No. 4,
OCTOBER
1990
251
Immunofluorescence assay
A pool of monoclonal antibodies to human influenza virus A nucleoprotein was kindly supplied by Dr. A. Kendal (CDC, Atlanta, Georgia USA) (8). On each smear, we deposited
20 microlites of monoclonal antibodies diluted 1: 20 in PBS pH 7.4. The slides were incubated in a humidified chamber at 37°C for 30 min, washed in PBS for 10 min and dried. The smears were then incubated with 20 microlitres of a 1: 100 dilution of the conjugate with Evans Blue as counter stain (0.04%) and examined with a Leitz Orthoplan microscope.
Downloaded by [University of Kent] at 08:59 08 December 2014
RESULTS
Clinical course of the experimental infection and virus isolation Three pigs presented a first-stage hyperthermia of about 41°C, 39.8°C and 41°C respectively for pigs no 2, 4 and 5. With these pigs, a second-stage hyperthermia followed. The influenza A H1N1 virus was detected in pigs no 1, 3, 4 and 5 by the first day post-infection. The samples collected seven days post-infection were negative (Table 1). Detection of influenza virus by the immunofluorescence technique
Viral antigens could be detected by indirect IF as soon as the first day postinfection in four pigs. The cytoplasm of infected cells showed more intense fluorescence than the nucleus, which seemed to contain less material than in samples collected at earlier times post-infection (Figure 1). At the seventh day post-infection positive cells were found only in one pig (pig no 3). In the cells collected from control pigs the cytoplasm was not fluorescent. Table 1.
Detection of influenza virus by immunofluorescence (IF).
PIG n°
2
1
IF
VI
39,2
-
N
39.8
P
39,5
+
P
39,5
.......
p
39,5
+++
P
40,1
39.4
...
P 39,4
-
P
40
P
40
.+4.
P
39,5
+
P
39,8
P
39.6 4-.4
N
39,3
-
N
N
39,4 +++4
N
39
N
IF
VI
-
N
++++
39,8
N
DPI
IF
VI
0
.-
N
39
-
N
39,3
1
'
P
40,7
-
N
41
2
-
N
39,2
.....
p
N
39,5
".
P
39,2
-
P
39.2
N
39,2
3 4
-
5
7
-
IF
:
VI
:
T +
++ +++
++++
252
1
IF
-
6
4
3
T
V1
T
T
IF
VI
-
N
39,5
P
41
-
"
T
P 39.1
P
39.7
P
39,5
39,8
p
39,7
39.9
N
39.5
'
Immunofluorescence Virus Isolation : Temperature : 1 to 5 fluorescent cells : 5 to 10 fluorescent cells : 10 to 15 fluorescent cells I over 15 fluorescent cells THE VETERINARY QUARTERLY, VOL. 12, No. 4, OCTOBER 1990
Sensitivity and specificity of the immunofluorescence technique
Downloaded by [University of Kent] at 08:59 08 December 2014
The sensitivity of the IF test relative to virus isolation was estimated to be 82% with 35 samples collected from five experimentally infected pigs. No specimens from the five control pigs were positive for influenza virus antigen.
4.6
a
Figure I.
a
Fluorescent cell. Cytoplasm is strongly and nucleus is faintly stained.
DISCUSSION
The diagnosis of influenza swine infection by detection of influenza virus NP antigen in nasal cells by the indirect immunofluorescence method requires a minimum number of infected swab cells. The presence of influenza virus antigen was confirmed by fluorescent granules observed in the epithelial cells, especially in ciliated epithelial cells with a distinct cylindric goblet shape. THE VETERINARY QUARTERLY. VOL.
12, No. 4,
OCTOBER 1990
253
Downloaded by [University of Kent] at 08:59 08 December 2014
We used monoclonal antibodies specific for conserved epitopes on NP antigens of human virus (8). A pool of at least two monoclonal antibodies specific for different regions of the viral proteins showed greater sensitivity in detecting the virus than the most sensitive individual antibody components (8). In previous studies, polyclonal sera used with an FITC technique detected influenza virus with higher sensitivity than did monoclonal antibodies (6). The monoclonal antibodies described in this study have been evaluated for direct detection of influenza viral antigens in clinical specimens. With indirect immunofluorescent antibody staining the Mabs showed sensitivities comparable to those obtained with polyclonal hyperimmune animal sera (3). A diagnosis of influenza virus infection in swine by the fluorescent antibody technique has been performed on lung tissue of dead animals (5). In this protocol, fluorescence was detected only if several lung sections on several animals were examined. The technique is successfully and routinely used in Belgian diagnostic laboratories (Castryck, personal communication). ACKNOWLEDGEMENTS
The authors thank Dr. M. Aymard of the University of Lyon for providing monoclonal antibodies. REFERENCES I. 2. 3.
4.
5. 6.
7.
Blaskovic D, Szanto J, Albrecht P, Sadecky E, Lackovic V. Demonstrations of swine influenza virus in pigs by the fluorescent antibody method. Acta Virol 1964; 8: 401-9. Gardner PS, Mc Qui Ilin J. Rapid virus diagnosis. Application of immunofluorescence. Butterworth and Co, 1984. Mc Qui Ilin J, Made ley CR, Kendal AP. Monoclonal antibodies for the rapid diagnosis of influenza A and B virus infections by immunofluorescence. The Lancet 1985; 911-4. Orstavik I, Grandien M, Halonen P, Arstela P, Mordhost CH, Hornsleth A, Papow-Kraupp T, Mc Quillin J, Gardner PS, Almeida J, Bricout F, Marques A. Viral diagnosis using the rapid immunofluorescence technique and epidemiological implications of acute respiratory infections among children in different European countries. Bull WHO 1984; 62 (2): 307-13. Pensaert M, Haesebrouck F, Castryck F. Diagnosis of influenza virus infections in swine by the fluorescent.antibody technique. Proceeding IPVS. Barcelone, August 1987; 215. Philips DJ, Galland GG, Reimer CB, Kendal AP. Evaluation of a solid phase immuno assay with fluorescein isothiocyanate conjugated heterogenous or monoclonal antibodies for identification of virus isolates with influenza virus as a model. J Clin Microbiol 1982; 15: 931-7. Tatino I, Suzuki S, Kawamura A, Kawashima H, Kusano N, Aoyama Y, Sugiura A, Akao Y,
Gikawa K, Homma N, Naito M. Diagnosis in Influenza by means of fluorescent antibody 8.
technique. Jap J Exp Med 1962; 32 (6): 531-59. Walls HH, Harmon MW, Slagle JJ, Stocksdale C, Dendar AP. Characterization and evaluation
of monoclonal antibodies developed for typing influenza A and influenza B viruses. J Clin Microbiol 1986; 23: 240-5.
254
THE VETERINARY QUARTERLY, VOL. 12, No. 4, OCTOBER 1990
Veterinary Quarterly Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tveq20
Diagnosis of swine influenza with an immunofluorescence technique using monoclonal antibodies a
a
a
M. Onno , A. Jestin , P. Vannier & C. Kaiser
b
a
Ministère de l'Agriculture , CNEVA , Station de Pathologie Porcine, B.P. n° 9, Ploufragan, 22440, France b
CNEVA, Laboratoire Central de Recherches Vétérinaires , Ministère de l'Agriculture , B.P. n° 67, Maisons, Alfort Cedex, 94703, France Published online: 01 Nov 2011.
To cite this article: M. Onno , A. Jestin , P. Vannier & C. Kaiser (1990) Diagnosis of swine influenza with an immunofluorescence technique using monoclonal antibodies, Veterinary Quarterly, 12:4, 251-254, DOI: 10.1080/01652176.1990.9694274 To link to this article: http://dx.doi.org/10.1080/01652176.1990.9694274
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sublicensing, systematic supply, or distribution in any form to anyone is expressly
Downloaded by [University of Kent] at 08:59 08 December 2014
forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Diagnosis of swine influenza with an immunofluorescence technique using monoclonal antibodies M. Onnol, A. Jestinl, P. Vannierl, C. Kaiser2
Downloaded by [University of Kent] at 08:59 08 December 2014
SUMMARY Direct diagnosis of swine influenza infection by an indirect immunofluorescence technique using anti-nucleoproteine monoclonal antibody was compared with virus isolation. Sw/4115/85. Clinical Five 8-week-old pigs were inoculated with 2 x 107 EID50 of strain A H1N1Sw/ signs developed in only three pigs. Antigen was detected in nasal epithelial cells obtained from
all animals the first day after inoculation; the antigen was detected in one pig 6 days after the infection. Fluorescence was present in the nucleus, nucleolus and cytoplasm of infected cells. The indirect immunofluorescence test was specific and as sensitive as virus isolation in embryonated eggs, allowing a rapid diagnosis that could be achieved within hours.
INTRODUCTION
The laboratory identification of swine influenza by virus isolation or serology is still rather time-consuming and most often allows only a retrospective diagnosis. We thought that the rapid detection tests and the reagents used for the diagnosis of influenza in humans would be suitable for the rapid diagnosis of influenza in swine.
The immunofluorescence (IF) technique is commonly used to make a rapid diagnosis of influenza infection by detecting specific viral antigens in the desquamated epithelial cells (2, 3, 4, 7). Blaskovic made an attempt to obtain an early diagnosis of influenza in experimentally infected pigs by using an indirect immunofluorescent test and polyclonal antibodies (1). Diagnosis of influenza virus infections in swine by the fluorescent antibody technique on lung tissue of dead animals was reported in 1987 by Pensaert (5). The production of adequate monoclonal antibodies would considerably improve
the IF test. Such assays have already been performed in humans (7) and the sensitivity of the indirect IF test using anti-nucleoproteine (NP) monoclonal antibodies has been found to be higher than that of virus isolation. We selected an indirect IF test with anti-NP monoclonal antibodies in order to detect both HINI and H3N2 infections in nasal swabs from experimentally infected pigs.
1
MATERIALS AND METHODS
Virus and experimental infection
The swine influenza A HIN, strain no 153 (ref. OMS Sw/4115/85) was inoculated intratracheally (107 EID50) in five 8-week-old-specific-pathogen-free Large-White pigs. Five control animals were inoculated with MEM medium. Nasal swabs were collected daily for seven days post-inoculation. An aliquot of the samples was inoculated in the amniotic cavity of embryonated eggs. I
2
Ministere de l'Agriculture, CNEVA, Station de Pathologie Porcine, B.P. n° 9, 22440 Ploufragan,
France. Ministère de l'Agriculture, CNEVA, Laboratoire Central de Recherches Vétérinaires, B.P. n° 67, 94703 Maisons, Alfort Cedex, France.
THE VETERINARY QUARTERLY, VOL. 12,
No. 4,
OCTOBER
1990
251
Immunofluorescence assay
A pool of monoclonal antibodies to human influenza virus A nucleoprotein was kindly supplied by Dr. A. Kendal (CDC, Atlanta, Georgia USA) (8). On each smear, we deposited
20 microlites of monoclonal antibodies diluted 1: 20 in PBS pH 7.4. The slides were incubated in a humidified chamber at 37°C for 30 min, washed in PBS for 10 min and dried. The smears were then incubated with 20 microlitres of a 1: 100 dilution of the conjugate with Evans Blue as counter stain (0.04%) and examined with a Leitz Orthoplan microscope.
Downloaded by [University of Kent] at 08:59 08 December 2014
RESULTS
Clinical course of the experimental infection and virus isolation Three pigs presented a first-stage hyperthermia of about 41°C, 39.8°C and 41°C respectively for pigs no 2, 4 and 5. With these pigs, a second-stage hyperthermia followed. The influenza A H1N1 virus was detected in pigs no 1, 3, 4 and 5 by the first day post-infection. The samples collected seven days post-infection were negative (Table 1). Detection of influenza virus by the immunofluorescence technique
Viral antigens could be detected by indirect IF as soon as the first day postinfection in four pigs. The cytoplasm of infected cells showed more intense fluorescence than the nucleus, which seemed to contain less material than in samples collected at earlier times post-infection (Figure 1). At the seventh day post-infection positive cells were found only in one pig (pig no 3). In the cells collected from control pigs the cytoplasm was not fluorescent. Table 1.
Detection of influenza virus by immunofluorescence (IF).
PIG n°
2
1
IF
VI
39,2
-
N
39.8
P
39,5
+
P
39,5
.......
p
39,5
+++
P
40,1
39.4
...
P 39,4
-
P
40
P
40
.+4.
P
39,5
+
P
39,8
P
39.6 4-.4
N
39,3
-
N
N
39,4 +++4
N
39
N
IF
VI
-
N
++++
39,8
N
DPI
IF
VI
0
.-
N
39
-
N
39,3
1
'
P
40,7
-
N
41
2
-
N
39,2
.....
p
N
39,5
".
P
39,2
-
P
39.2
N
39,2
3 4
-
5
7
-
IF
:
VI
:
T +
++ +++
++++
252
1
IF
-
6
4
3
T
V1
T
T
IF
VI
-
N
39,5
P
41
-
"
T
P 39.1
P
39.7
P
39,5
39,8
p
39,7
39.9
N
39.5
'
Immunofluorescence Virus Isolation : Temperature : 1 to 5 fluorescent cells : 5 to 10 fluorescent cells : 10 to 15 fluorescent cells I over 15 fluorescent cells THE VETERINARY QUARTERLY, VOL. 12, No. 4, OCTOBER 1990
Sensitivity and specificity of the immunofluorescence technique
Downloaded by [University of Kent] at 08:59 08 December 2014
The sensitivity of the IF test relative to virus isolation was estimated to be 82% with 35 samples collected from five experimentally infected pigs. No specimens from the five control pigs were positive for influenza virus antigen.
4.6
a
Figure I.
a
Fluorescent cell. Cytoplasm is strongly and nucleus is faintly stained.
DISCUSSION
The diagnosis of influenza swine infection by detection of influenza virus NP antigen in nasal cells by the indirect immunofluorescence method requires a minimum number of infected swab cells. The presence of influenza virus antigen was confirmed by fluorescent granules observed in the epithelial cells, especially in ciliated epithelial cells with a distinct cylindric goblet shape. THE VETERINARY QUARTERLY. VOL.
12, No. 4,
OCTOBER 1990
253
Downloaded by [University of Kent] at 08:59 08 December 2014
We used monoclonal antibodies specific for conserved epitopes on NP antigens of human virus (8). A pool of at least two monoclonal antibodies specific for different regions of the viral proteins showed greater sensitivity in detecting the virus than the most sensitive individual antibody components (8). In previous studies, polyclonal sera used with an FITC technique detected influenza virus with higher sensitivity than did monoclonal antibodies (6). The monoclonal antibodies described in this study have been evaluated for direct detection of influenza viral antigens in clinical specimens. With indirect immunofluorescent antibody staining the Mabs showed sensitivities comparable to those obtained with polyclonal hyperimmune animal sera (3). A diagnosis of influenza virus infection in swine by the fluorescent antibody technique has been performed on lung tissue of dead animals (5). In this protocol, fluorescence was detected only if several lung sections on several animals were examined. The technique is successfully and routinely used in Belgian diagnostic laboratories (Castryck, personal communication). ACKNOWLEDGEMENTS
The authors thank Dr. M. Aymard of the University of Lyon for providing monoclonal antibodies. REFERENCES I. 2. 3.
4.
5. 6.
7.
Blaskovic D, Szanto J, Albrecht P, Sadecky E, Lackovic V. Demonstrations of swine influenza virus in pigs by the fluorescent antibody method. Acta Virol 1964; 8: 401-9. Gardner PS, Mc Qui Ilin J. Rapid virus diagnosis. Application of immunofluorescence. Butterworth and Co, 1984. Mc Qui Ilin J, Made ley CR, Kendal AP. Monoclonal antibodies for the rapid diagnosis of influenza A and B virus infections by immunofluorescence. The Lancet 1985; 911-4. Orstavik I, Grandien M, Halonen P, Arstela P, Mordhost CH, Hornsleth A, Papow-Kraupp T, Mc Quillin J, Gardner PS, Almeida J, Bricout F, Marques A. Viral diagnosis using the rapid immunofluorescence technique and epidemiological implications of acute respiratory infections among children in different European countries. Bull WHO 1984; 62 (2): 307-13. Pensaert M, Haesebrouck F, Castryck F. Diagnosis of influenza virus infections in swine by the fluorescent.antibody technique. Proceeding IPVS. Barcelone, August 1987; 215. Philips DJ, Galland GG, Reimer CB, Kendal AP. Evaluation of a solid phase immuno assay with fluorescein isothiocyanate conjugated heterogenous or monoclonal antibodies for identification of virus isolates with influenza virus as a model. J Clin Microbiol 1982; 15: 931-7. Tatino I, Suzuki S, Kawamura A, Kawashima H, Kusano N, Aoyama Y, Sugiura A, Akao Y,
Gikawa K, Homma N, Naito M. Diagnosis in Influenza by means of fluorescent antibody 8.
technique. Jap J Exp Med 1962; 32 (6): 531-59. Walls HH, Harmon MW, Slagle JJ, Stocksdale C, Dendar AP. Characterization and evaluation
of monoclonal antibodies developed for typing influenza A and influenza B viruses. J Clin Microbiol 1986; 23: 240-5.
254
THE VETERINARY QUARTERLY, VOL. 12, No. 4, OCTOBER 1990
