AVIAN DISEASES 53:491–494, 2009
Serosurvey and Diagnostic Application of Antibody Titers to Aspergillus in Avian Species Carolyn Cray,AC Toshiba Watson,A and Kristopher L. ArheartB A
University of Miami Miller School of Medicine, Division of Comparative Pathology, P.O. Box 016960 R-46, Miami, FL 33101 B Department of Epidemiology and Public Health, P.O. Box 016960 R-669, Miami, FL 33101 Received 2 March 2009; Accepted and published ahead of print 23 May 2009 SUMMARY. A multiyear study was conducted using an enzyme-linked immunosorbent assay to measure antibody to address the application of the test to the diagnosis of aspergillosis in avian species. In general serostudies (n 5 1314), four avian groups (psittaciform, raptor, penguin, and zoo) were found to have samples with antibody reactivity. Penguin, raptor, and zoo groups were found to have higher levels of antibody to Aspergillus than the psittaciform group. Additional clinical information was collected on 303 cases, which resulted in the definition of presumptive normal, probable, and confirmed infection groups. Although the confirmed group was more likely to have antibody reactivity, the mean antibody index was not found to be significant between presumptive normal and probable or confirmed cases.
RESUMEN. Muestreo serolo´gico y aplicacio´n diagno´stica de los tı´tulos de anticuerpos para Aspergillus en especies aviares. Se condujo un estudio que abarco´ varios an˜os utilizando un ensayo de inmunoabsorcio´n con enzimas ligadas para medir anticuerpos y evaluar la aplicacio´n de este procedimiento en el diagno´stico de aspergilosis en especies aviares. En los estudios serolo´gicos generales (n 5 1314), cuatro grupos aviares (psita´cidos, aves rapaces, pingu¨inos y aves de zoolo´gico) mostraron tener reaccio´n de anticuerpos. Los grupos de pingu¨inos, aves rapaces y aves de zoolo´gico mostraron tener los tı´tulos ma´s altos de anticuerpos para Aspergillus en comparacio´n con el grupo de psita´cidos. Se recolecto´ informacio´n clı´nica adicional de 303 casos, lo que ayudo´ a definir grupos de aves presuntamente normales, con infeccio´n probable y con infeccio´n confirmada. Aunque el grupo confirmado era el que tenı´a la mayor probabilidad de mostrar reactividad de anticuerpos, los promedios de niveles de anticuerpos no fueron significativamente diferentes entre los casos presuntamente normales, probables y confirmados. Key words: aspergillosis, antibody, psittaciform bird, Aspergillus Abbreviations: ELISA 5 enzyme-linked immunosorbent assay; PBS 5 phosphate-buffered saline
Aspergillosis is a major cause of morbidity and mortality in avian species. Antemortem diagnosis remains problematic. While routine hematology and chemistry analyses, culture, and radiography have value, they do not provide a definitive diagnosis (19). Endoscopy can be a good antemortem tool but is not always available or possible due to the advanced stage of disease in patients. Practitioners often depend on routine testing such as a leukocytosis of an unknown origin as well as clinical signs to form a diagnosis and initiate treatment. Serodiagnostic testing for aspergillosis was first described in ducks and pigeons (5,22). In 1994, Redig and colleagues described the first application of an antibody enzyme-linked immunosorbent assay (ELISA) in companion birds and raptors (4). Many studies have documented the high prevalence of antibodies in captive and wild penguins (13,14,25). Zielezienski-Roberts and Cray reported the use of antibody testing in conjunction with detection of circulating Aspergillus antigen and changes in protein electrophoresis (10,29). The goal of the current study was twofold. First, a large serosurvey was conducted to assess the prevalence of antibody reactivity to Aspergillus in different avian species using the ELISA technique. Second, using clinically defined groups, the application of antibody titers to the diagnosis of aspergillosis was assessed. MATERIALS AND METHODS Study samples. Lithium heparinized plasma samples were submitted to the University of Miami Avian and Wildlife Laboratory from 2004 C
Corresponding author. E-mail:
[email protected]
through 2006 for analysis by an Aspergillus antibody ELISA. All samples were analyzed within 24 hr of arrival at the laboratory. All samples were refrigerated at 4 C until analyzed. A total of 1314 samples were analyzed. This includes 886 samples from psittaciform species, 104 samples from raptor species (Orders Falconiformes and Strigiformes; predominated by eagles, hawks, owls, and vultures), 108 samples from various penguin species (Order Sphenisciformes), and 216 samples from zoologic institutions predominated by land and waterfowl species (Orders Anseriformes and Galliformes including chickens, ducks, geese, peafowl, pheasants, swans, turkeys, and quail). Fewer samples in the latter groups were also analyzed from the following orders: Columbiformes, Galliformes, Charadiformes, Passeriformes, Caprimulgiformes, Coraciiformes, Musophagiformes, Pelecaniformes, and Ciconiiformes. The psittaciform group was predominated by African grey parrots (n 5 183), macaws (n 5 220), cockatoos (n 5 147), conures (n 5 36), eclectus parrots (n 5 48), and Amazon parrots (n 5 110). These random samples were analyzed without information regarding clinical condition or diagnosis. Defining clinical groups. Throughout the study period, submitting practitioners were sent patient questionnaires. This included collecting information such as whether there was clinical confirmation of infection (by histology), clinical signs, accessory diagnostic testing (i.e., radiography, hematology, chemistry), and whether the patient responded to treatment. Three groups were defined: presumptive normal (nonaspergillosis, n 5 70), confirmed (histologic confirmation, n 5 57), and probable (cumulative data and observations strongly consistent with aspergillosis, n 5 176). Greater than 85% of each clinical group was psittaciform birds. Antibody assay. An indirect ELISA was adapted from techniques described in previous reports (13,24). Unfractionated Aspergillus antigen (Immuno-mycologics, Norman, OK) was coated on Nunc Maxisorp Immunomodule ELISA plates (Rochester, NY) overnight at 4 C. After a wash with phosphate-buffered saline (PBS), the plate was blocked with
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Table 1. Aspergillus antibody reactivity by avian groups. Group
% Cases $ 1.4
% Cases , 1.4
Psittaciform n 5 886 Zoo species n 5 216 Raptor n 5 104 Penguin n 5 108
32A 86B 93 98
68 14 7 2
Mean index 6 SE
1.22 1.85 1.88 2.01
6 6 6 6
0.01A 0.03C 0.04D 0.04
A
P , 0.001 vs. all other groups. P 5 0.004 vs. penguin group. C P 5 0.001 vs. penguin group. D P 5 0.023 vs. penguin group. B
10% casein solution (Sigma, St. Louis, MO) for 2 hr at room temperature. After PBS wash, the plate was stored at 4 C until use. Plasma samples were diluted with PBS and 50 ml was plated per well. After a 30-min incubation at 37 C, the plate was washed with a saline 0.05% Tween 20 solution three times. Horseradish peroxidase conjugated anti-chicken IgG (Sigma) was added to each well for 30 min at 37 C. The anti-chicken IgG antibody has previously been described as cross reactive with other avian species (8). After salineTween washes, the substrate 2,29-azino-bis(3-ethylbenzthiazoline-6sulphonic acid (Kirkegaard and Perry, Gaithersburg, MD) was added. The plate was read at 405 nm using a Molecular Devices plate reader (Sunnyvale, CA). Positive and negative control samples were run simultaneously with the test samples. The absorbance of the negative controls was averaged. Test sample absorbance was divided by the negative control average to produce an index. For all reagents, the ELISA was optimized using a checkerboard titration technique using known positive and negative samples. Statistical analysis. An index of 1.4 was selected as a cutoff for purposes of data analysis. Previously, we determined that the index of 1.4 provided maximum sensitivity and specificity for this antibody ELISA (29). Logistic regression was used to analyze the qualitative antibody data for the bird groups, parrot species, and the clinical groups. A one-way analysis of variance with planned contrasts was used to determine if the assay indices were significantly different among the bird groups, parrot species, and the clinical groups.
RESULTS
Serosurvey. Four groups of birds were analyzed for antibody reactivity to Aspergillus. A significantly higher number of zoo, penguin, and raptor species were found to have an antibody index greater than 1.4 vs. psittaciform birds ( P , 0.001 for all comparisons); indices of penguin species were significantly higher than waterfowl ( P 5 0.004; Table 1). More than 90% of penguin and raptor samples demonstrated antibody reactivity with an index greater than 1.4. The average antibody index was significantly lower in the psittaciform vs. the other groups ( P , 0.001 for all comparisons); zoo species and raptors were significantly lower than penguins ( P 5 0.001 and P 5 0.023, respectively). The psittaciform bird group was further analyzed by species (data not shown). The macaw group had a significantly higher proportion with an antibody index greater that 1.4 than any of the other parrot species (African gray parrots P 5 0.003; conures P , 0.001; cockatoos P , 0.001; eclectus parrots P 5 0.010; and Amazon parrots P 5 0.014). African gray parrots had a significantly higher proportion of results greater than 1.4 than conures ( P 5 0.011) and cockatoos ( P 5 0.004), and Amazon parrots had a significantly higher proportion of results greater than 1.4 than conures ( P 5 0.011) and cockatoos ( P 5 0.008). The same significance pattern was observed for the average antibody index. Clinical group analysis. Clinical groups were defined as presumptive normal, probable infection, and confirmed infection.
Fig. 1. Percentage of cases with antibody indices ,1.4 and $1.4 on the basis of clinical grouping: presumptive normal, probable, and confirmed.
Qualitative evaluation of antibody reactivity is presented in Fig. 1. Whereas the indices of 69% of presumptive normal birds and 60% of the probable infection birds were less than 1.4, only 42% the confirmed group were below this index. The differences between the percentages of the presumptive normal and the other two groups was significant ( P 5 0.003 and P 5 0.022, respectively). However, when the actual values were analyzed, there was a not a significant difference between the presumptive normal and the confirmed group ( P 5 0.059; Table 2). DISCUSSION
Aspergillosis is a major cause of mortality in avian species. In a review of aspergillosis in animal species, Tell indicated that birds may have a predilection for aspergillosis due to their lack of epiglottis, diaphragm, and surface macrophages as well as a warm body temperature that may promote fungal growth (27). In a survey of different avian groups without regard to clinical status, this study has shown high antibody prevalence among raptor, zoo, and penguin species. Approximately 94% of the raptor samples were seropositive. This is in contrast to reports by Redig using a similar assay but with different species conjugate antibodies and may reflect a difference in assay sensitivity (4). Specific data on waterfowl species have not been reported, but an experimental model of infection found that Pekin ducks readily seroconverted after challenge (15). Aspergillosis in Galliformes has been well described (11). The positive serostatus of penguin species has also been well characterized in the literature (13,14,25). This finding was reproduced in the current study, and the highest mean reactivity index was also observed in this species. The difference between the results observed in penguins, raptors, and zoo species vs. the psittaciform bird group may reflect repeated antigenic challenges due to increased exposure that would be concomitant with housing, husbandry, and environment that is Table 2. Mean and standard error of index results for antibody reactivity in the Aspergillus ELISA by clinical group. An analysis of variance was conducted with planned contrasts to compare probable and confirmed with normal. Clinical Group
Antibody Index
Normal (n 5 70) Probable (n 5 176) Confirmed (n 5 57)
1.26 6 0.05 1.35 6 0.03A 1.41 6 0.06B
A B
P 5 0.131 vs. normal group. P 5 0.059 vs. normal group.
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unique to the aforementioned three groups. Penguins may also be genetically predisposed to being high responders to Aspergillus since even wild penguins are often seropositive (14). To this point, Reidarson and colleagues suggested that all penguins may be colonized with Aspergillus (25). In the psittaciform group; the lower reactivity may reflect a lower exposure to antigen and perhaps a lower overall incidence of disease. A second factor is the possible limited immune responsiveness of this population due to stress, nutrition, and secondary diseases. The finding of higher antibody reactivity in macaws is interesting, but without epidemiologic documentation of a different incidence of disease in this species, it remains not understood. On the basis of experimental models of infection in mice and continued studies in humans, it has been proposed that cellmediated immunity, especially TH1 type responses, is important in later stages of the disease and that an excessive humoral immune response may result in the progression of disease (3,26). In contrast, antibody may provide a form of resistance to initial infection together with physical barriers, phagocytes, and other parts of the innate immune system (3). Previously, Graczyk et al. reported, in an experimental model of infection of Pekin ducks, that antibody titers did not correlate with histopathology or changes in blood parameters including white blood counts and total protein (15). Studies using ELISA techniques to monitor samples from naturally infected penguins resulted in the same conclusion (13). In the current study, when antibody reactivity was examined in light of clinical context using a defined cutoff point of 1.4, it is notable that although the mean index by clinical group was not found to be statistically significant, there was a clear difference in the percentage of antibody positive birds in the confirmed group vs. the presumptive normal group. The probable and confirmed clinical groups were heavily weighted by the presence of weak positive reactivity on the ELISA. This suggests that any result over an index of 1.4 may be considered potentially reflective of infection. However, since each clinical group was composed of greater than 85% psittaciform birds, the data in the current study may be weighted by this one order of birds. Future studies considering data of documented infection in raptors, penguins, and various zoo species may necessitate a reevaluation of the index cutoff used to define positive vs. negative antibody reactivity. That is, different cutoff indices may be used with different orders of avian species to increase the specificity and sensitivity of the assay. Differences in antibody reactivity may also be dictated by the stage of infection. To this point, the low levels of antibody reactivity may be present due to the immune suppressive properties of Aspergillus toxins including gliotoxin (20). This premise is further supported by presence of 42% of samples with antibody indices less than 1.4 in the confirmed group. In total, these data are consistent with those reported previously in a study of aspergillosis in seven psittaciform birds (17). The current study demonstrates only a mixed association of the presence of antibody and aspergillosis in avian species. This is similar to information collected in nonavian species with a variety of techniques including ELISA, immunoblot, and immunodiffusion. Billen et al. recently reported low concentrations of antibody in normal control dogs although significantly higher reactivity could be observed in dogs with confirmed sino-nasal aspergillosis (2). The utility of antibody testing in dogs was also confirmed by others (12). Anti-Aspergillus antibody has also been detected in normal cattle and horses (16,18). In horses, it was reported to be difficult to differentiate between healthy and infected animals, although testing by immunoblot to different individual fungal antigens improved the utility of the assay (16). In our laboratory, studies are underway to
address this possibility using antigen specific ELISA and Western blot techniques. Testing for antibody reactivity may be further enhanced through the use of other diagnostic tests including hematology, radiology, and endoscopy (19). The value of antigen testing has also been reported in a model of experimental infection in Pekin ducks (15). A commercial assay to measure galactomannan, a major antigen of Aspergillus, has been widely applied in human medicine (23). A recent publication profiled its application to infection in falcons with a high specificity but low sensitivity (1). Additionally, Le Loc’h and colleagues reported a higher incidence of positive galactomannan results in psittaciform birds, although there were a significant number of false positive results (21). Our laboratory has also recently demonstrated its use in the diagnosis of aspergillosis in avian species (6,9). Protein electrophoresis has been reported to be a reflection of acute inflammation and stimulation of humoral immunity in avian species (7,28). While not diagnostic of aspergillosis, the positive application of electrophoresis has been reported in confirmed cases in psittaciform birds (9,17). The inclusion of cases of probable or presumptive infection in this study was preceded by studies of clinical aspergillosis in humans (23). As with avian medicine, there is a segment of a patient population that is consistent with infection and responds to treatment. This prevents the confirmation of infection that would be obtained with further invasive diagnostics and autopsy. This epidemiologic factor of patient definition clearly produced different results from the confirmed group, although very similar result trends and significance from the presumptive normal group were observed. It could be inferred from the current study that the confirmed group, since patients were confirmed at necropsy, may represent birds that were tested during a more advanced stage of infection. Likewise, those cases in the probable group may represent birds with clinical signalment and diagnostic analysis that allowed for an earlier treatment intervention. It is important to note that the current study included birds with focal and generalized infection, although all appeared to be a chronic presentation of disease. As the database of clinical presentation, specific sites of infection, and prognostic use grows, the evaluation of antibody reactivity will need to be continuously validated for further application to the aspergillosis diagnostic dilemma. REFERENCES 1. Arca-Ruibal, B., U. Wernery, R. Zachariah, T. A. Bailey, A. Di Somma, C. Silvanose, and P. McKinney. Assessment of a commercial sandwich ELISA in the diagnosis of aspergillosis in falcons. Vet. Rec. 158:442–444. 2006. 2. Billen, F., D. Peeters, I. R. Peters, C. R. Helps, P. Huynen, P. De Mol, L. Massart, M. J. Day, and C. Clercx. Comparison of the value of measurement of serum galactomannan and Aspergillus-specific antibodies in the diagnosis of canine sino-nasal aspergillosis. Vet. Microbiol. 133:358–365. 2009. 3. Blanco, J. L., and M. E. Garcia. Immune response to fungal infections. Vet. Immunol. Immunopathol. 125:47–70. 2008. 4. Brown, P. A., and P. T. Redig. Aspergillus ELISA: a tool for detection and management. Proc. Annu. Conf. Assoc. Avian Vet. 295–297. 1994. 5. Buxton, I., and C. V. Sommer. Serodiagnosis of Aspergillus fumigatus antibody in migratory ducks. Avian Dis. 24:446–454. 1980. 6. Cray, C., D. R. Reavill, A. Romagnano, F. Van Sant, D. Champagne, R. Stevenson, V. Rolfe, C. Griffin, and S. Clubb. Galactomannan assay and protein electrophoresis findings in twelve psittaciform birds with aspergillosis. J. Avian Med. Surg 23:125–135. 2009. 7. Cray, C., and L. M. Tatum. Applications of protein electrophoresis in avian diagnostics. J. Avian Med. Surg. 12:4–10. 1998. 8. Cray, C., and D. Villar. Cross-reactivity of anti-chicken IgY antibody with immunoglobulins of exotic avian species. Vet. Clin. Pathol. 37:328– 331. 2008.
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