Mycopathologia 140: 121–127, 1998. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.
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Value of antigen and antibody detection, and blood evaluation parameters in diagnosis of avian invasive aspergillosis Thaddeus K. Graczyk1,2 , Michael R. Cranfield2,3 & Patrice N. Klein3,4 1 Department
of Molecular Microbiology and Immunology, School of Hygiene and Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, Maryland 21205, USA 2 Medical Department, The Baltimore Zoo, Druid Hill Park, Baltimore, Maryland 21217, USA 3 Division of Comparative Medicine, School of Medicine, Johns Hopkins University, 720 Rutland Avenue, Baltimore, Maryland 21205, USA 4 The Humane Society of the United States, 2100 L Street N.W., Washington, DC 20037, USA. Received 29 July 1997; accepted in final form 10 March 1998
Abstract The applicability of ELISA detection of circulating Aspergillus spp. antigen (Ag) and systemic antibody (Ab) of IgG class, and the blood parameter values were evaluated for diagnosis of invasive aspergillosis in Aspergillus spp.challenged Peking ducks (Anas platyrhynchos). The protective role of Aspergillus spp. IgG was evaluated in Cape shelducks (Tadorna cana) immunized with Aspergillus spp. Ag. Challenged but non-immunized A. platyrhynchos developed invasive aspergillosis on day 21 as demonstrated histopathologically by the presence of fungal microgranuloma in air sacs and lung tissue, with serum antigenemia fluctuating from 65 to 270 ng of 55-kD basic protein Ag per ml. Immunized A. platyrhynchos did not demonstrate Aspergillus spp. serum antigenemia but did show rare histopathological changes in some air sacs associated with fungal inflammation. Although the differences between immunized and non-immunized T. cana in blood evaluation parameters did not differ significantly, immunized birds mounted high Aspergillus spp.-specific IgG titer. There was no correlation between the blood parameter values and post-immunization timepoints in T. cana and in A. platyrhynchos. Intramuscular immunization with Aspergillus spp. mycelial phase cultures Ag provided protection against the pathogens. The lack of relations between blood parameter values and increasing Aspergillus spp. IgG titers (in T. cana and A. platyrhynchos) indicate low applicability of these parameters in evaluation of a bird Aspergillus spp. status. Detection of circulating 55-kDa Aspergillus spp. Ag has high early predictive values for invasive aspergillosis in birds. Key words: Aspergillus spp., Aspergillus antigen, avian aspergillosis, circulating antigen, inhibition ELISA. Abbreviations: Ab, antibody; Ag, antigen; ELISA, enzyme-linked immunosorbent assay
Introduction The fungi, Aspergillus fumigatus, A. niger, and A. flavus cause significant mortality in avian hosts [1,2,3,4]. Although aspergillosis is one of the most important air-borne infections of captive birds maintained in indoor exhibitions [5] and represents a serious threat to poultry health [6], the studies on bird immunoresponses to the pathogen are scant. Humoral responses of IgG type of Domestic turkeys (Meleagridis gallopava) exposed to A. fumigatus spores
were detected by enzyme-linked immunosorbent assay (ELISA) [7], and IgM and IgG responses were demonstrated in pigeons (Columba livia) immunized with A. fumigatus culture filtrate extract [8]. The magnitude of antibody (Ab) responses of M. gallopava were correlated to the number of A. fumigatus spores inhaled by the birds, and based on Ab titer it was possible to distinguish subclinical and clinical infections in experimentally infected turkeys [7]. Captive African black-footed penguins (Spheniscus demersus) mounted systemic IgG responses against Aspergillus
122 spp. as demonstrated by ELISA utilizing 55-kDa basic protein Aspergillus spp. antigen (Ag) [9]. Aspergillus spp.-specific IgG were concentrated in the penguin egg yolk, and detected in the hatchling serum [9]. Immunodiagnosis of aspergillosis based on systemic Ab is complicated by the fact that these fungi are widespread and exposure of birds to these pathogens is common [10]. Detection of circulating Aspergillus Ag is an advanced technique to diagnose invasive aspergillosis in immunosuppressed or immunocompromised humans whose ability to mount Ab responses were impaired [11]. Although it has been demonstrated that birds mount Aspergillus-specific humoral responses, it remains unknown if it is possible to detect circulating Ag of Aspergillus in avian sera. The purpose of the present study was to determine the value of detection of systemic Aspergillus spp.specific IgG and circulating Aspergillus spp. Ag in immunodiagnosis of avian invasive aspergillosis, and to assess if induced by immunization Aspergillus spp.specific IgG responses can provide protection against these pathogens. In addition, we tested applicability of blood and serum chemistry parameters for diagnosis of bird infection with Aspergillus spp.
Materials and methods Twelve (5 males, 7 females) adult (1-yr-old), captivereared, and housed outdoor Cape shelducks (Tadorna cana) from the Baltimore Zoo (Baltimore, Maryland, USA) were used. Six birds (3 males and 3 females) were immunized, and 6 non-immunized birds, injected with saline solution served as controls. The immunization suspension, 1 ml, delivered intramuscularly (i.m.) on day 1, 14, and 35, was prepared from a purified, alcohol precipitated, mycelial phase cultures of A. fumigatus, A. niger, and A. flavus of carbohydrate concentration of 0.77 mg/ml [9]. Carbohydrate suspension was mixed with equal volume of Freund’s complete adjuvant. Intramuscular delivery was chosen because intravenous immunization of birds against A. fuminatus has been ineffective in generating immunoresponses [8]. Blood samples of 2.0 ml volume were collected from all ducks on day 1, 7, 14, 21, 28, 35, 42, and 49 post-immunization (PI) [9]. Leucocyte evaluation was carried out for total white blood cells (WBC) counts (× 103 /µl), segmented heterophils (HETS) (per µl), and lymphocytes (LYMPHS) (per µl). Erythrocyte evaluation included red blood cell (RBC)
counts (× 106/µl) and hematocrit (HCT) (%). In addition a serum chemistry parameter, total protein (PROT) (µm/dl) was determined. Leucocyte and erythrocyte evaluations, and serum chemistry analysis were carried out as described previously [12,13]. Eighteen, 2-day-old Peking ducks (Anas platyrhynchos) purchased from a commercial hatchery (The Ridway Hatcheries, Inc., La Rue, Ohio, USA), were divided at the age of 4 wk, while housed indoor, into 3 groups of 5 birds (Group A, B, and C), and Group D of 3 birds. The Groups of A and C were housed in separate rooms, and Group B and D were housed in the same room. The ducks from Group B and Group C were administered 3.5 × 104 of Aspergillus spp. spores each via catheter directly to the trachea, and birds from Group C were immunized i.m. on day 7, 14, 21, 28, and 35 post-challenge. Immunization suspension was prepared as described above and production of the spores followed previously described procedures [7]. The birds from Group A and Group D served as controls; these birds were not challenged with Aspergillus spp. and were not immunized. Blood samples of 1.0 ml were collected from all birds twice at 1 wk interval prior to challenge with Aspergillus spp., and then on day 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, 28, and 35 post-challenge. Leucocyte and erythrocyte evaluations, and serum chemistry analysis were carried out as for the Cape shelducks. All Peking ducks were euthanized on day 35 post-challenge by overexposure to saturated CO2 atmosphere and necropsied. Trachea, lung, and airsac tissues were collected and preserved in 10% phosphate buffered formalin. The tissue samples were embedded in paraffin, 5-µm-thick sections stained with Hematoxylin and Eosine (H + E), and Methenamine Silver Gomori-Grocott stain (MS), and examined by routine light microscopy. A direct enzyme-linked immunosorbent assay (ELISA) was carried out [14] to determine the binding efficacy of alkaline phosphatase-labelled anti-duck IgG (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Maryland, USA) to IgG in the T. cana and A. platyrhynchos sera. All T. cana and A. platyrhynchos serum samples were processed by the indirect ELISA to detect anti-Aspergillus spp. IgG. Optimum sensitivity was achieved using Immulon 2 microtitration plates (Dynatech Laboratories, Inc., Chantilly, Virginia, USA). The wells were coated in triplicate with 100 µl (5 Fg/ml) of 55-kDa basic protein Aspergillus spp. Ag [9]. The 55-kDa Ag was prepared by removing cell
123 wall galactoman and glucan from a purified, alcohol precipitated, mycelial phase cultures of A. fumigatus, A. niger, and A. flavus using Concanavalin ASepharose 4B affinity column [9]. The 55-kDa Ag was electroeluted from the SDS-PAGE gel [9]. The protocol of ELISA followed previous procedures [9]. The negative control (NC) serum was obtained by precipitation of the immunoglobulins and immunocomplexes [11] from a pool of serum samples of T. cana and A. platyrhynchos. Positive control (PC) serum was obtained from two Mallard ducks (A. platyrhynchos) that died due to clinical aspergillosis. Positive and NC sera were prepared at the same time, frozen at −70 ◦ C in small amount in eppendorf tubes, and a single samples were thawed for the ELISA then discharged after use. This procedure prevented changes in the ELISA background caused by repeated freeze-thaw cycles. The positive cutoff level was established as unit of Ab greater than the mean of absorbance ± 3 SD of NC serum. All serum samples of T. cana and A. platyrhynchos were processed by the inhibition ELISA to determine Aspergillus spp. antigenemia. For inhibition ELISA, the wells on Immulon 2 microtitration plates were coated in triplicate with 100 µl (5 Fg/ml) of 55-kDa basic protein Aspergillus spp. Ag [9] and incubated at 42 ◦ C for 3 h. Following coating the plate was postcoated by incubating each well with 200 µl of casein blocking buffer/0.5% TW-20 [14]. The wells were emptied, air dried and stored at −70 ◦ C until needed. Immunoglobulins and immunocomplexes from the tested serum samples were coagulated [11] and the samples were incubated 1.0 h at 42 ◦ C with 1/5 volume of the PC serum. The incubate was used to fill in triplicate ELISA plate wells (100 µl each) previously coated with Aspergillus spp. Ag, and the remaining procedure followed the protocol for the indirect ELISA. Standard curve, generated by adding Aspergillus spp. Ag to the NC serum at concentrations of 13, 25, 50, 100, 200, 400, 800, and 1,600 ng/ml, was assayed on each inhibition ELISA plate. Sensitivity of the Aspergillus spp. antigenemia assay was determined as described previously [11]. Eight wells on each indirect and inhibition ELISA plates were not coated with Ag to determine nonspecific background values (NBV) of absorbance. The absorbance values from various ELISA plates were compared as described previously [15]. Absorbance values were read at 405 nm on a MAXline microplate reader (Molecular Devices Corp., Menlo Park, California, USA) controlled by a computer program (SOFTmax, Molecular
Devices Corp., Menlo Park, California) that subtracted NBV from all absorbance values on the plate, giving the corrected absorbance values. Statistical analysis was carried out with the Analytical Software Statistix 4.1 (Analytical Software, St. Paul, Minnesota, USA). Analysis of variance (ANOVA) was carried out to determine the significance of the among-group effect, and two-sample t- test was applied to compare the mean values. Mean values (x) were associated with standard deviation (SD).
Results The concentration of 5 µg/ml of 55-kDa basic protein Aspergillus spp. Ag, and a dilution of 1/500 of alkaline phosphatase-labelled anti-duck IgG was optimal in terms of sensitivity and specificity of the indirect ELISA. The range of absorbance values for PC serum was 0.78 to 0.84; x = 0.81 ± 0.02. The NC serum gave a range of absorbance, 0.10 to 0.12; x = 0.11 ± 0.01, and the cutoff level at 0.14. The lowest concentration values of 55-kDa basic protein Aspergillus spp. antigenemia obtained in the inhibition ELISA standard curves varied within the limits of 57–75 ng/ml with the Ag detection sensitivity of 65 ng/ml. Analysis of variance showed that differences between immunized and nonimmunized T. cana in all parameters were not significant (ANOVA; F = 21.2, P > 0.05). Also, values of the blood evaluation and serum chemistry parameters did not differ significantly among A. platyrhynchos groups (F = 21.2, P > 0.05). In T. cana and in A. platyrhynchos there was no significant correlation between parameter values and subsequent PI or post-challenge/immunization timepoints, respectively. Also, differences among male and female ducks in T. cana groups, and between males and females within each T. cana group, were not significant (ANOVA; F = 16.6, P > 0.05). All T. cana were positive for Aspergillus spp. IgG prior to immunization. The range of absorbance was 0.21–0.42, x = 0.33 ± 0.02 for control ducks, and 0.30–0.45, x = 0.38 ± 0.03 for ducks designed for immunization. Prior to immunization, the differences in the mean Ab titer between these T. cana groups were not significant (two-sample t-test; t = 3.23, P > 0.05). All immunized T. cana generated Aspergillus spp.specific IgG responses (Table 1). Two ducks showed significantly elevated IgG level a week after immunization (two-sample t-test; t = 3.1, P < 0.02). When compared with the baseline values, IgG titer of all im-
124 munized T. cana was significantly elevated 2 weeks PI (two-sample t-test; t = 2.7, P < 0.01). Interestingly, Aspergillus spp. IgG kinetic profiles of these 6 immunized ducks varied significantly (ANOVA; F = 3.1, P < 0.05) among birds. No Aspergillus spp. Ag was detected in T. cana sera. No histopathological changes were observed in birds in the control groups. The most pronounced inflammatory changes reflecting fungal invasion were seen in the airsacs and in lung tissue of ducks that received intratracheal inoculation of spores and were not immunized (Group B). Three of 5 ducks had mild granulomatous airsacculitis with scattered presence of fungal fruiting bodies, spores, and hyphal structures which stained positive by MS. Lung tissue sections of 2 of these ducks had mild granulomatous pneumonia with positively stained (MS) fungal elements, and mild multifocal lymphoid hyperplasia. In contrast, 2 of 5 ducks that received intratracheal inoculation of spores and were immunized (Group C) had only occasional microgranulomas containing few fungal elements in the airsac tissues. In this group, all lung tissues were negative for fungal invasion. No significant histopathological changes were seen in the tracheal tissue from any A. platyrhynchos in this study. All A. platyrhynchos were negative for Aspergillus spp. IgG prior to the challenge. Seven days postchallenge, Aspergillus spp.-specific IgG were detected in 1 A. platyrhynchos and, all Group C and B birds had detectable IgG starting on day 14 PI (Table 2). The mean IgG titer of nonimmunized vs immunized A. platyrhynchos challenged with pathogen spores was not significantly different (two-sample t-test; t = 2.7, P = 0.05). However, the kinetic profiles differ significantly between these 2 groups of ducks (ANOVA; F = 2.8, P < 0.03); Ab titers of immunized ducks increased more rapidly than in non-immunized birds. The kinetic profiles ofAspergillus spp. 55-kDa basic protein antigenemia of the serum of 3 A. platyrhynchos challenged with the pathogen spores and nonimmunized is presented in Figure 1. The Ag was detected in the sera starting from day 21 post-challenge. Concentration of detected Ag fluctuated within the range of 65 to 270 ng/ml over the time (Figure 1). These 3 birds had also the highest Aspergillus spp. IgG titer; however, the blood and serum parameter values were within the range of the control birds (Table 1).
Figure 1. Concentration of circulating Aspergillus spp. antigen in the serum of three captive Peking ducks (Anas platyrhynchos) (represented by triangle, circle, and square) that developed invasive aspergillosis after experimental intratracheal challenge with 3.5 × 104 Aspergillus spp. spores.
Discussion The potential cross-reactivity between Aspergillus spp. and Mucor spp., Rhizopus spp., Penicillium spp., and Candida spp.-derived protein antigen(s) (Ag) was excluded previously [16,17]. We consider detection of anti-Aspergillus spp. IgG utilizing 55-kDa basic protein Ag of Aspergillus spp. to be genus-specific. Thus, the indirect ELISA showed that all T. cana housed outdoors were exposed to the pathogen prior to immunization. This is not unexpected as the pathogen spores are commonly present in the indoor [18] and outdoor habitats [3,19]. The present study supported results obtained for experimental infection of domestic turkeys with A. fumigatus [7] that the presence of systemic Aspergillus spp.-specific IgG and their titer can be useful in diagnosis of invasive aspergillosis in birds. However, because Aspergillus spp. expresses extremely broad spectrum of cross-reactive Ag during infection, and due to the lack of standardized Ag, applicability of ELISA in immunodiagnosis of invasive aspergillosis in birds is limited. The present study demonstrated that a 55-kDa basic protein Aspergillus spp. Ag is circulating in the serum of infected birds during invasive aspergillosis, and it is possible to detect this Ag by inhibition ELISA. We conclude that detection of serum 55-kDa Ag has important early predictive values and antigen-
125 Table 1. Relative titers of systemic Aspergillus spp.-specific IgG, and the values of blood and serum chemistry parameters of captive Cape shelducks (Tadorna cana) intramuscularly immunized with Aspergillus spp. mycelial phase culture antigen Parametersa
Immunized ducks Mean SD Range
Nonimmunized ducks Mean SD Range
IgG titer WBC (103 /µl) RBC (106 /µl) HCT (%) HETS (per µl) LYMPHS (per µl) TPROT (g/dl)
0.67 10.3 4.0 50.8 34.2 61.8 5.0
0.33 12.2 3.7 48.9 33.7 63.3 4.9
0.7 1.5 0.2 1.8 1.5 7.6 0.4
0.56–0.77 8.4–12.2 3.7–4.2 49.0–53.0 32.5–36.7 46.6–66.6 4.9–5.1
0.02 3.3 0.2 2.7 8.4 7.1 0.2
0.21–0.42 9.2–18.4 3.5–4.0 44.7–51.3 23.0–45.1 53.4–72.3 4.7–5.2
a IgG titer – determined by the indirect enzyme-linked immunosorbent assay and absorbance values read at 405 nm wavelength, WBC – white blood cells, RBC – red blood cells, HCT – hematocrit, HETS – segmented heterophils, LYMPHS – lymphocytes, and TPROT – total protein.
Table 2. Relative titers of systemic Aspergillus spp.-specific IgG, concentration of circulating Aspergillus spp. antigen, and the values of blood and serum chemistry evaluation parameters of captive Peking ducks (Anas platyrhynchos) that were challenged with 3.5 × 104 Aspergillus spp. spores and were (or were not) intramuscularly immunized with Aspergillus spp. mycelial phase culture antigen Parametersa
IgG titer Ag (ng/ml) WBC (103 /µl) RBC (106 /µl) HCT (%) HETS (per µl) LYMPHS (per µl) TPROT (g/dl)
Control Mean SD
Range
Infected and nonimmunized Mean SD Range
Infected and immunized Mean Sd Range
0.37 0 19.1 2.1 29.7 57.6 27.5 4.1
0.22–0.49 0 8.9–23.8 1.9–3.7 25.2–39.1 51.0–85.5 13.4–34.7 3.9–4.3
0.78 131 18.5 2.2 30.9 59.9 25.5 4.0
0.82 0 20.8 2.1 31.4 58.1 27.2 4.1
0.03 0 1.7 0.3 3.6 5.7 0.4 0.5
0.08 16.7 1.9 0.3 3.6 5.1 0.3 0.5
0.42–0.98 65–270 5.9–27.8 1.8–3.3 26.2–37.1 49.0–82.5 11.4–32.7 3.8–4.4
0.09 0 2.5 0.2 4.0 6.0 2.9 0.4
0.57–1.05 0 10.5–40.3 2.0–2.3 28.7–35.0 48.6–80.7 18.8–37.8 3.9–4.2
a IgG titer – determined by the indirect ELISA, Ag - 55-kDa basic protein Aspergillus spp. antigen detected by inhibition
ELISA, WBC – white blood cells, RBC – red blood cells, HCT – hematocrit, HETS - segmented heterophils, LYMPHS – lymphocytes, and TPROT - total protein.
emia can be used for diagnosis of Aspergillus spp. infection in birds before the clinical onset of the invasive disease. Captive birds mount IgG responses against this Ag, and these IgG, highly concentrated in the egg-yolk are passed to the hatchlings [9]. The Ag used in the present study can be easily produced in large amounts that would facilitate standardization of the Ag and Ab detection ELISA. Considering the fact that several avian conjugates are commercially available, these assays can be applicable to other groups of birds. The use of 55-kDa basic protein Ag offers twofold advantages as the indirect ELISA for detection of systemic Ab, and inhibition ELISA for detection of circulating Ag, can be carried out simultaneously. Due
to the difficulties in finding Aspergillus spp.-negative avian serum, we recommend generating a negative control for ELISA by coagulation of immunoglobulins and immunocomplexes from available avian serum. The essential component of the Aspergillus spp. Ag detection based on inhibition ELISA is the high-IgG titer detector serum. The serum can be obtained from bird(s) with clinical aspergillosis. For assay standardization purposes, we recommend storing detector serum frozen at −20 ◦ C or −70 ◦ C in small aliqoutes, e.g., 0.5 ml, and do not re-refreeze the serum after use. The sensitivity of Aspergillus spp. Ag detection in mammalian sera by ELISA varies from 1 ng/ml to 68 ng/ml depending on type of immunore-
126 agents [3,20,21]. Fluctuations of the antigenemia levels demonstrated in the A. platyrhynchos sera conform to high changes in Aspergillus spp. Ag concentrations observed in animal experimental models with immunocompetent and immunosuppressed rabbits [20], and in serum of immunosuppressed humans [11]. For clinical decisions however, far more important than high assay sensitivity, is knowledge what Ag concentration is indicative of invasive disease. Based on the results of the present study, we conclude that concentrations of Aspergillus spp. Ag of 65 ng/ml, or higher, are highly indicative of invasive aspergillosis in birds. Histopathological changes found in A. platyrhynchos that displayed serum antigenemia (Group B) were consistent with the pathologic changes associated with invasive aspergillosis seen in domestic turkeys [7]. In contrast, only 2 of 5 ducks that were Aspergillus spp.-challenged and immunized (Group C) showed occasional Aspergillus spp.-related histopathological changes to a lesser extend than Group B, and were negative for circulating Ag. These results suggest that i.m. immunization against Aspergillus spp. provided protection, and although the birds were exposed to pathogen and strongly seropositive, the disease did not develop. When the vaccine protection was not provided, as in nonimmunized ducks that were challenged with the spores, a 3 week period was long enough for development of invasive aspergillosis in naive birds. Domestic turkeys that inhaled Aspergillus spp. spores developed invasive aspergillosis within 18 to 21 days [7]. The present study showed that in the 2 groups of A. platyrhynchos, both of which were challenged with the pathogen spores, but only one group was immunized, both had similar high Aspergillus spp.-specific IgG titer. Although the nonimmunized ducks had more extensive and pronounced histopathological changes consistent with invasive aspergillosis than the immunized ducks, based on IgG titer these 2 groups were indistinguishable. The predominant strength of the immunodiagnostic approach proposed in the present study is that an individual serum sample can be assayed simultaneously for Aspergillus spp.-specific IgG and Aspergillus spp. Ag. Intramuscular immunization of T. cana that had pre-existing Aspergillus spp.-specific IgG titers induced great elevation of the titer. Based on the results obtained from A. platyrhynchos, it can be assumed that immunization provided protection against the pathogens in T. cana. Unfortunately, the increase of Aspergillus spp.-specific IgG titers did not correlate
with blood evaluation and serum chemistry parameter values either in T. cana nor A. platyrhynchos indicating that the values of these parameters in evaluation of Aspergillus spp. status of a bird are low.
Acknowledgements We thank S. Sarro, D. Heyl, S. Maltese, S. Davis, and L. Campbell for technical assistance, and J. Strandberg (Johns Hopkins University, Baltimore, Maryland, USA) for facilitating the study. This study was supported by the Maryland Zoological Society, and the AKC Fund of New York.
References 1.
2.
3.
4.
5.
6.
7.
8.
9.
10. 11.
12.
13.
Bair WC, Simpson SG, Windigstad RM. Acute aspergillosis in mallards at Oahe Seep Near Pierre, South Dakota. Prairie Nat 1988; 20: 153–156. Brandt CJ, Windigstad RM, Siegfried LM, Duncann RM, Cook RM. Avian morbidity and mortality from botulism, aspergillosis, salmonellosis at Jamaica Bay Wildlife Refuge, New York, USA. Colon Waterbirds 1988; 11: 284–292. Singh H, Grewal GS, Singh N. Mycotic salpingitis in a Japanese quail (Coturnis coturnis japonica). Avian Dis 1994; 38: 910–913. Redig PT. Avian aspergillosis. In: Fowler ME, ed. Zoo and wild animals medicine. W. B. Saunders Company, Philadelphia, 1993; 178–181. Stoskopf MK. Penguin and alcid medicine. In: Fowler ME, ed. Zoo and wild animals medicine, W. B. Saunders Company, Philadelphia, Pennsylvania, 1993; 189–194. Beckman BJ, Howe CW, Trampel DW, DeBey MC, Richard JL, Niyo Y. Aspergillus fumigatus keratitis with intraocular invasion in 15-day-old chicks. Avian Dis 1994; 38: 660–665. Redig PT, Post GS, Concannon TM, Dunette J. Development of an ELISA for the detection of aspergillosis in avian species. Proc Assoc Avian Vet 1986; 165–178. Martinez-Quesada J, Nieto-Cadenazzi A, Torres-Rodriquez JM. Humoral immunoresponse of pigeons to Aspergillus fumigatus antigen. Mycopathologia 1993; 124: 131–137. Graczyk TK, Cranfield MR. Maternal transfer of anti- Aspergillus spp. immunoglobulins in African black-footed penguins (Spheniscus demersus). J Wildl Dis 1995; 31: 545–549. Graczyk TK, Cockrem JF. Aspergillus spp. seropositivity in New Zealand penguins. Mycopathologia 1995; 131: 179–184. Johnson TM, Kurup VP, A. Resnick A, Ash RC, Fink JN. Detection of circulating Aspergillus fumigatus antigen in bone marrow transplant patients. J Lab Clin Med 1989; 114: 700– 707. Graczyk TK, Cranfield MR, Bicknese EJ. Evaluation of serum chemistry values associated with avian malaria infections in African black-footed penguins (Spheniscus demersus). Parasitol Res 1995; 81: 316–319. Graczyk TK, Shaw ML, Cranfield MR, Beall FB. Hematologic characteristics of avian malaria cases in African black- footed penguins (Spheniscus demersus) during the first outdoor exposure season. J Parasitol 1994; 80: 302–308
127 14. Graczyk TK, Cranfield MK, Skjoldager ML, Shaw ML. An ELISA for detecting anti-Plasmodium spp. antibodies in African black-footed penguins (Spheniscus demersus). J Parasitol 1994; 80: 60–66. 15. Schwartz BS, Ford DP, Childs JE, Rothman N, Thomas RJ. Anti- tick saliva antibody: a biologic marker of tick exposure that is a risk factor for Lyme disease seropositivity. Am J Epidemiol 1991; 134: 86–95. 16. Redig PT, Cooper JE, Remple JD, Hunter DB, eds. Raptor biomedicine. University of Minnesota Press, Minneapolis, 1993. 17. Carrasco L, Bautista MJ, de las Mulas JM, Jensen HE. Application of enzyme-immunochemistry for the diagnosis of aspergillosis, candidiasis, and zygomycosis in three lovebirds. Avian Dis 1993; 37: 923–927. 18. Lentino JR, Rosekranz MA, Michaels JA, Kurup VP, Rose DH, Rytel MW. Nosocomial aspergillosis; a retrospective review of airborne disease secondary to road construction and contaminated air conditioners. Am J Epidemiol 1982; 116: 430–437.
19.
20.
21.
Mercantini R, Marsella R, Cervellati MC. Keratinophilic fungi isolated from Antarctic soil. Mycopathologia 1989; 106: 47– 52. Patterson TF, Miniter P, Ryan JL, Andriole VT. Effect of immunosuppression and amphotericin B on Aspergillus antigenemia in an experimental model. J Inf Dis 1988; 158: 415–422. Rohrlich P, Sarfati J, Mariani P, Duval M, Carol A, SaintMartin C, Bingen E, Latge JP, Vilmer E. Prospective sandwich enzyme-linked immunosorbent assay for serum galactomannan: early predictive value and clinical use in invasive aspergillosis. Ped Inf Dis J 1996; 15: 232–237.
Address for correspondence: Dr. Thaddeus K. Graczyk, The Johns Hopkins University, School of Hygiene and Public Health, Department of Molecular Microbiology and Immunology, 615 North Wolfe Street, Baltimore, Maryland 21205, USA. Phone: (410) 614-4984, Fax: (410) 955-0105, Rm 4307A E-mail:
[email protected]
