ELISA appears to be a less reliable method than counterimmunoelectrophoresis for the diagnosis of nasal aspergillosis in the dog. False-positive or ...
Sabouraudia: Journal of Medical and Veterinary Mycology (1984) 22, 251-254
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Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Aspergillus fumigatus intranasal infection of the dog Z. U. K H A N *~, M. D. R I C H A R D S O N ~, D. W. W A R N O C K ~, AND J. G. LANE 2
1Department of Microbiology, Bristol Royal Infirmary, Bristol BS2 8HW, and 2Department of Veterinary Surgery, University of Bristol, Langford House, Bristol BS18 7DU, England (Accepted 2 November 1983)
ELISA appears to be a less reliable method than counterimmunoelectrophoresis for the diagnosis of nasal aspergillosis in the dog. False-positive or false-negative results were recorded for anti-A. fumigatus IgG in nine animals with aspergiUosis and in 27 disease-free dogs although this problem could be reduced with careful selection of antigen.
Aspergillusfumigatus infection of the nasal and frontal sinuses of the dog is a well recognized condition which must be considered in all cases where a mesocephalic or dolichocephalic animal is presented with intermittent or persistent unilateral or bilateral nasal discharge or epistaxis [1, 3, 4]. Differentiation of this infection from other canine disorders with otherwise similar clinical and radiological presentations is often difficult. Direct smears and culture of nasal discharge are not reliable [2, 3, 4]. Indeed, isolation of A. fumigatus from nasal tissue often necessitates surgical intervention. Typical radiographic changes are often seen which may be helpful in the diagnosis [4]. Where these findings are not available or confirmation is required, precipitin tests have been shown to be a reliable diagnostic method [3, 5, 9]. The object of this investigation was to assess the usefulness of ELISA for diagnosis of nasal aspergillosis in the dog. Four commercial antigens were used. Antigens 1 and 2 were prepared from liquid cultures of A. fumigatus incubated for varying lengths of time as either surface or submerged growth (Bencard, Brentford, England). Antigens 3 and 4 were somatic and culture filtrate preparations (Mercia Brocades Limited, West Byfleet, England). Antigens 5 and 6 were culture filtrate preparations made from 3-week-old cultures of A. fumigatus in glucose-asparagine broth. Nine dogs with nasal aspergillosis were studied: all had haemorrhagic, purulent, *Present address: Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110007, India. Correspondence address: Dr M. D. Richardson, Department of Microbiology, Bristol Royal Infirmary, Bristol BS2 8HW, England. 251
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unilateral or bilateral, intermittent or persistent nasal discharge, together with radiological signs of destructive rhinitis. Nasal cultures were not performed, but all nine animals had precipitins in counterimmunoelectrophoresis (CIE) tests with A. fumiga. tus antigens, performed according to the method of Richardson et al. [7]. Three dogs had precipitins against all six antigens, one dog had precipitins against five antigens, and one against 4 antigens. Serum was also obtained from 18 dogs with nasal discharge and from nine normal dogs. None of these 27 animals had precipitins to A. fumigatus. The indirect ELISA used for the determination of anti-A, fumigatus IgG was based on the method of Richardson et al. [6] with horseradish peroxidase-conjugated rabbit antiserum to canine IgG (Miles Laboratories, Stoke Poges, England). The levels of anti-A, fumigatus IgG detected in the 36 dogs studied are shown as absorbance values (490 nm) in Fig. 1. Most sera from the 27 dogs without aspergillosis had values lower than the minimum recorded for the nine dogs with aspergillosis. Sera from the nine normal dogs produced minimal absorbance values in tests with some antigens, but gave much higher values (maximum absorbance value of 0.25) in tests with three culture filtrate preparations (antigens 4-6). 1.0 ANTIGEN 1
ANTIGEN 3
ANTIGEN 2
0.8
0.6
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1.0 ANTIGIEN 4
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ANTIGEN 6
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c~oops FIG. 1. Distribution of absorbance values in ELISA for anti-A.fumigatus IgG in sera from dogs with: nasal aspergillosis (Group 1); or nasal discharge (group 2); and normal animals (Group 3). ( O CIE testnegative; O CIE test-positive).
For each antigen in Fig. 1, the dogs with aspergillosis have been recorded as CIEpositive or -negative. In most cases, high absorbance values in ELISA correlated with positive reactions in CIE with the same antigen. However, in a number of cases,
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higher values were r e c o r d e d in E L I S A w h e r e no p r e c i p i t i n s h a d b e e n d e t e c t e d in C I E with the s a m e antigen. In an a t t e m p t to d i s c r i m i n a t e b e t w e e n positive a n d n e g a t i v e sera in E L I S A [8], o n e positive reference s e r u m ( f r o m a d o g w i t h n a s a l aspergillosis w i t h strong p r e c i p i t i n s ) and 10 pools o f n e g a t i v e reference sera ( f r o m 30 dogs with n a s a l d i s c h a r g e b u t n o precipitins) were i n c l u d e d in e a c h test run. T h e a b s o r b a n c e v a l u e o f e a c h s e r u m was then expressed as E L I S A units: as a p e r c e n t a g e o f t h e a b s o r b a n c e v a l u e o f the positive reference serum. Test sera were r e g a r d e d as positive i f t h e i r E L I S A unit v a l u e was greater t h a n the m e a n E L I S A unit v a l u e p l u s 2 S D o f the 10 n e g a t i v e r e f e r e n c e pools. The n u m b e r o f dogs t h a t were r e c o r d e d as E L I S A - p o s i t i v e for e a c h A. fumigatus antigen is listed in T a b l e 1. TABLE 1. Correlation between CIE and ELISA for IgG antibodies to A. fumigatus in 36 dogs
Antigen 1 2 3 4 5 6
Number of dogs giving positive reactions in ELISA: Nasal Nasal Reaction aspergillosis discharge Normal in CIE (9) (18) (9) Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative
4~ 3~ 6 2h 7 2 8 1 7 2 3 6
-1 -0 -3 -2 -6 -6
-0 -0 -0 -0 -0 -0
° One dog positive in CIE with this antigen, but negative in ELISA. b One dog negative in CIE and ELISA with this antigen. The n u m b e r o f dogs w i t h aspergillosis r e c o r d e d as positive in E L I S A r a n g e d f r o m 7 to 9 a c c o r d i n g to the a n t i g e n used. T h e highest n u m b e r o f n e g a t i v e results r e c o r d e d in this group o f a n i m a l s was t w o w i t h a n t i g e n 1. N o n e g a t i v e results were r e c o r d e d w i t h four o f the six antigens. T h e n u m b e r o f dogs w i t h o u t aspergillosis r e c o r d e d as positive in E L I S A r a n g e d f r o m 0 to 6. T h e highest n u m b e r o f positive results in this g r o u p o f 27 C I E - n e g a t i v e a n i m a l s was r e c o r d e d w i t h the c u l t u r e filtrate p r e p a r a t i o n s ( a n t i g e n s 4-6). It has b e e n f o u n d t h a t the choice o f positive a n d n e g a t i v e r e f e r e n c e sera for inclusion in E L I S A c a n h a v e a m a r k e d influence o n the n u m b e r o f positive results obtained [7]. I n this investigation, n e g a t i v e reference sera were o b t a i n e d f r o m 30 dogs with nasal d i s c h a r g e a n d w i t h o u t A. fumigatus precipitins. E v e n t h o u g h these sera produced m i n i m a l a b s o r b a n c e v a l u e s in E L I S A w i t h the d i f f e r e n t A. fumigatus antigens, positive results were still o b t a i n e d a m o n g the g r o u p o f 18 s i m i l a r test dogs with n a s a l d i s c h a r g e b u t n o precipitins. Previous i n v e s t i g a t i o n s h a v e d e m o n s t r a t e d t h a t the d e t e c t i o n o f A. fumigatus precipitins is a r e l i a b l e m e t h o d for the d i a g n o s i s o f n a s a l aspergillosis in dogs, since n o false-positive o r f a l s e - n e g a t i v e results were r e c o r d e d [3, 5]. E L I S A a p p e a r s to be a much less r e l i a b l e m e t h o d for d i a g n o s i s o f this c o n d i t i o n b e c a u s e o f the n u m b e r o f false-positive a n d false-negative results t h a t were o b t a i n e d . It d o e s a p p e a r t h a t this
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p r o b l e m c a n b e r e d u c e d , i f n o t e l i m i n a t e d , t h r o u g h c a r e f u l c h o i c e o f a n t i g e n preparation. E L I S A does, h o w e v e r , p r o v i d e a m e a n s o f d e t e c t i n g l o w l e v e l s o f i m m u n o g l o b u lin t h a t c a n n o t b e d e m o n s t r a t e d w i t h p r e c i p i t i n m e t h o d s . M o r e o v e r , it p e r m i t s detailed i n v e s t i g a t i o n o f d i f f e r e n t i m m u n o g l o b u l i n classes o f a n t i b o d i e s to A. furnigatus in dogs (or o t h e r a n i m a l s ) w i t h a s p e r g i l l o s i s . ZUSAMMENFASSUNG Fiir die Diagnose von nasaler Aspergillose des Hundes scheint Elisa eine weniger zuved~issige Methode zu sein als Counterimmunoelektrophorese. Mit einem Anti-A. fumigatus IgG wurden falsch positive oder falsch negative Resultate bei 9 Tieren mit Aspergillose und bei 27 gesunden Hunden beobachtet. Diese Schwierigkeit kotmte vermindert werden dutch eine sorgfaltige Auswahl des Antigens. ACKNOWLEDGEMENT One of us (ZUK) was awarded a fellowship under the scientific exchange programme between the Indian National Science Academy and the Royal Society (London). REFERENCES 1. BARRETT,R. E., HOFFER, R. E. ~¢ SCHULTZ,R. D. 1977. Treatment and immunological evaluation of three cases of canine aspergillosis. Journal of the American Animal Hospital Association, 13, 328-334. 2. CHANDLER, E. A. 1975. Aspergillus infection of the nasal cavity of the dog: diagnosis and treatment. Veterinary Record, 96, 136. 3. LANE, J. G. & WARNOCK,D. W. 1977. The diagnosis of Aspergillus fumigatus infection of the nasal chambers of the dog with particular reference to the value of the double diffusion test. Journal of Small Animal Practice, 18, 169-177. 4. LANE, J. G., CLAYTON-JONES,D. G., THODAY, K. L. & THOMSETT, L. R. 1974. The diagnosis and successful treatment of Aspergillusfumigatus infection of the frontal sinuses and nasal chambers of the dog. Journal of Small Animal Practice, 15, 79-87. 5. POLl, G., PONTI, W., BALSARI,A., ADDIS, F. & MORTELLARO,C. M. 1981. Aspergillusfumigatus and specific precipitins in dogs with turbinate changes. Veterinary Record, 108, 143-145. 6. RICHARDSON,M. D., STUBSINS,J. M. & WARNOCK,D. W. 1982. Rapid enzyme-linked immunosorbent assay (ELISA) for Aspergillusfumigatus antibodies. Journal of Clinical Pathology, 35, 1134-1137. 7. RICHARDSON,M. D., SMITH, S. M. • WARNOCK,D. W. 1983. Further evaluation of factors affecting a rapid ELISA procedure for detection of IgG antibodies to Candida albicans. Mycopathologia, 82, 167-173. 8. RICHARDSON,M. D., TURNER,A., WARNOCK,D. W. & LLEWELLYN,P. A. 1983. Compnter-assisted rapid enzyme-linked immunosorbent assay (ELISA) in the serological diagnosis of aspergillosis. Journal of Immunological Methods, 56, 201-207. 9. RICHARDSON,M. D., WARNOCK,D. W., BOVEY,S. E. & LANE,J. G. 1982. Rapid serological diagnosis of Aspergillus fumigatus infection of the frontal sinuses and nasal chambers of the dog. Research in Veterinary Science, 33, 167-169.
