Immunologic study of systemic aspergillosis in German Shepherd dogs

Veterinary Immunology and lmmunopathology, 9 (1985) 3 3 5 - - 3 4 7 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

335

IMMUNOLOGIC STUDY OF SYSTEMIC ASPERGILLOSIS IN GERMAN SHEPHERD DOGS M.J. DAY1, C.E. EGER2, S.E. SHAW2 AND W.J. PENHALE1 i D i v i s i o n of Veterinary Biology 2Division of Applied Veterinary Medicine School of Veterinary Studies, Murdoch University, Murdoch, Western Australia, 6150 (Australia) (Accepted 12 March 1985) ABSTRACT Day, M.J., Eger, C.E., Shaw, S.E. and Penhale, W.J., 1985. Immunologic study of systemic a s p e r g i l l o s i s in German Shepherd dogs. Vet. Immunol. Immunopathol., 9: 335-347. Data are presented from a series of eight cases of disseminated canine a s p e r g i l l o s i s (A. terreus) in German Shepherd dogs referred to Murdoch University Veterinary Hospital. Immunoglobulin determination revealed depression of serum IgA (cases i and 5) and IgM (case 2) levels and elevated levels of IgG in a l l cases. Total complement a c t i v i t y (CHs0) and complement components tested, (C3, C4) were present in normal amounts in a l l cases. Using agar gel d i f f u s i o n , serum antibody to A. t e r r e u s was found in only one case and aspergillus antigenaemia in two of the remainder. Lectin transformation of lymphocytes in two dogs was found to be depressed r e l a t i v e to normal controls in case 1 and i n i t i a l l y in case 2. Two dogs f a i l e d to respond to the intradermal injection of A. t e r r e u s antigen. INTRODUCTION Aspergillus species have been reported as the cause of localized or disseminated mycotic infections in birds, domestic livestock (Soltys and SumnerSmith, 1971), cats (McCausland, 1971), horses, rabbits (Black and Nightingale, 1973) and man (Seligsohn et a l . , 1977; Laham and Carpenter, 1982). Aspergillosis due to A. f u m i g a t u s s e l e c t i v e l y involving the nasal c a v i t i e s and paranasal sinuses has been reported in dogs of predominantly dolichocepha l i c breeds (Barrett et a l . , 1977). discharge, sneezing and dyspnoea.

Major presenting signs include nasal On radiographic examination turbinate

destruction is present (Lane and Warnock, 1977). Systemic a s p e r g i l l o s i s caused by A. t e r r e u s has been reported on a number of occasions in man (Seligsohn et a l . , 1977; Laham and Carpenter, 1982) and recently there have been isolated reports of i t s occurrence in the dog (Wood et a l . , 1978; Weitkamp, 1982; Mullaney et a l . , 1983). Aspergillus terreus is a saprophytic fungus normally found in soil and plant matter (Raper and Fennell, 1965).

A study by Rippon et al (1971) showed

that c l i n i c a l l y isolated strains of A. t e r r e u 8 had a greater virulence, metabo l i c rate and growth rate than soil isolates. 0165-2427/85/$03.30

In compromised human hosts the

© 1 9 8 5 Elsevier Science Publishers B.V.

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organism has been described as causing skeletal and cardio-pulmonary pathology (Seligsohn et a l . , 1977; Laham and Carpenter, 1982). During the period 1980-1984 eight cases of disseminated a s p e r g i l l o s i s in adult German Shepherd dogs have been presented to Murdoch U n i v e r s i t y Veterinary Hospital (M.U.V.H.).

These dogs were referred in the terminal stages of

the disease and had a common h i s t o r y of chronic pyrexia, anorexia, uraemia, weight loss and lameness. This study examines the c l i n i c o - p a t h o l o g i c a l f i n d i n g s and general and s p e c i f i c immunological status of these dogs. MATERIALS AND METHODS Eight cases of systemic A. t e r r e u s

i n f e c t i o n were referred to M.U.V.H.

from Perth metropolitan p r a c t i t i o n e r s with e i t h e r terminal disease or for necropsy examination.

Suitable samples f o r immunological study were obtained

from f i v e of these animals. Humoral immunity was assessed by serum protein electrophoresis, immunog l o b u l i n q u a n t i t a t i o n and agar gel d i f f u s i o n to detect serum antibody to A.

terreus.

Complement involvement was measured using t o t a l haemolytic com-

plement (CHs0) and serum concentrations of components C3 and C4 by single radial immunodiffusion. Cell mediated immunity was evaluated using intradermal skin t e s t i n g and lymphocyte transformation by phytohaemagglutinin (PHA) and A. t e r r e u s

antigen.

The polymorphic complement C4 immunogenetic marker was used to study genetic predisposition. Serum Protein Electrophoresis (SPE) SPE was performed on agarose gels using the Corning electrophoresis system. Immunoglobulin Determinations Immunoglobulin determinations were performed using commercially prepared antibody impregnated agarose radial immunodiffusion plates I for canine IgG, IgM and IgA.

Known standard sera included in these k i t s were used to construct

reference curves f o r the test samples. Serum Antibody to A. terreus A s t r a i n of A . t e r r e u s

isolated from one of these cases was used to pre-

pare antigen as described by Poll et al (1981).

Fungal mycelia were recovered

from Czapek-Dox medium, washed and f i n e l y ground in saline with a mortar.

This

suspension was shaken f o r 48 hours before being centrifuged at 3500 rpm f o r 30 minutes.

The r e s u l t i n g supernatant was spectrophotometricaliy assayed for

337

protein content ( i mg/ml) and concentrated 100 f o l d f o r use in gel d i f f u s i o n s . A. t e r r e u s

antigen was tested by agarose gel d i f f u s i o n against ~erum from

the f i v e t e s t dogs and twenty four randomly selected normal German Shepherd controls. Serum C3 and C4 Quantitation Serum concentrations of complement components C3 and C4 were determined using a single radial immunodiffusion technique as described by Day et a l , (1984).

Four percent antiserum to canine C32 or C43 was incorporated into

agarose plates and ring diameter was measured 48 hours a f t e r loading 5ul t e s t serum samples. The squared p r e c i p i t a t i o n diameters obtained using sera from I00 random control dogs were plotted on a frequency histogram.

The midpoint of the

modal segment of these histograms was assigned a value of I00 a r b i t r a r y u n i t s and a serum containing t h i s amount of C3 or C4 d i l u t e d to 75, 50 and 25 u n i t s to produce a reference curve.

The cumulative frequencies of serum concentrat-

ions of C3 and C4 in the control populations were plotted and the 95th perc e n t i l e used to define normal levels of each component. Total Haemolytic Complement Complement CHs0 values were determined by t i t r a t i o n .

B r i e f l y , sheep red

blood c e l l s (SRBC), sensitized by incubating with a previously determined concentration of r a b b i t anti SRBC antibody, were suspended in glucose complement working buffer (0.075 m o l a r i t y , pH7.2) at 5% v/v. 0.5 ml of serial d i l u t i o n s of test serum were incubated at 37°C f o r 90 minutes with 0.5 ml of sensitized SRBC and 1.5 ml of complement working b u f f e r . Tubes were subsequently centrifuged at I000 g f o r 10 minutes and absorbance measured at 541 nm in a spectrophotometer.

0 and 100% l y s i s control tubes

were included. A curve of percentage l y s i s versus the log of reciprocal serum d i l u t i o n was plotted on l o g / l i n e a r paper f o r each sample from which the CHs0 value could be d i r e c t l y obtained. Lymphocyte Blastogenesis C e l l u l a r immune function was assessed by l e c t i n s t i m u l a t i o n of lymphocyte or whole blood cultures.

B r i e f l y , heparinized blood or separated lymphocytes

(4 x 105 per w e l l ) were incubated in m i c r o t i t r e trays f o r 3 days at 37°C in IMiles Laboratories, Springvale, V i c t o r i a 2Ra~bitanti-dog C3, Miles Laboratories, Springvale, V i c t o r i a 3Goat anti-dog C4, Lawrence Laboratories, Western A u s t r a l i a

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an atmosphere of 5% C02. Isolated lymphocytes were cultured in a mixture of RPMI medium with 5% foetal calf serum and 2 mercapto-ethanol (4 x 10-5M) incorporating doses of phytohaemagglutinin (PHA-P) from 10-40 mg/ml. Cultures were pulsed over the final 6 hours with 3H thymidine 4 (0.5 uCi/ culture well).

The lymphocytes were washed on a glass f i l t e r with phosphate

buffered saline (PBS, pH 7.6, 0.01 M) using a Titertek cell harvester.

Whole

blood cultures were treated with a series of d i s t i l l e d water and alcohol washes to lyse erythrocytes. The f i l t e r s were placed in Beckman Redi-Solv s c i n t i l l a t i o n cocktail and counted in a Searle Isocap 300 s c i n t i l l a t i o n counter.

Results were expressed

as transformation indices which were determined by calculating the ratio of counts per minute (CPM) of stimulated to non-stimulated samples. Intradermal Skin Testing 0.05 ml quantities of mixed Aspergillus antigen (Commonwealth Serum Laboratories, 1000 PNU/ml), A. terreus antigen, a positive control (histamine, 28 ~g/ ml) and a negative control ( s t e r i l e saline) were injected intradermally into a prepared site on the flank of cases 1 and 5. Skin reactions at injection sites were assessed q u a l i t a t i v e l y at 15-20 minutes and again at 24 hours post injection. Complement C4 Allotyping C4 allotype was determined using the method described by Kay and Dawkins (1984).

A neuraminidase treated EDTAplasma sample was electrophoretically

separated in agarose and C4 bands immunofixed by overlying with antiserum to canine C4. RESULTS Case Studies A summary of the c l i n i c a l , laboratory and pathological findings from the cases is presented in Tables I, I I and I l l . The c l i n i c a l signs shown in these cases were referable to the anatomical sites of the fungal granulomas. Consistently, the musculoskeletal system was involved and where vertebral osteomyelitis occurred, neurological dysfunction was evident.

In the l a t t e r stages of the disease c l i n i c a l signs

referable to pyelonephritis and/or renal f a i l u r e were common. In 4 of the 8 cases, uveitis or endophthalmitis preceded more generalized signs of illness by several months. .4New England Nuclear, N.S.W.

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TABLE I Clinical and Radiographic Findings Age (Yrs)

Sex

3

2

Case

Presenting Signs

Radiographic Findings

M

Paraparesis. Lameness l e f t foreleg; swollen l e f t carpus. Endophthalmitis l e f t eye. Weight loss, pyrexia, generalized weakness.

Discospondylitis involving T2-T4 and TT-T8 vertebrae. Destructive lesion of l e f t radial carpal bone.

3

F

Lameness r i g h t foreleg; Discospondylitis swollen r i g h t humeroscapu- Ts-T6, TI0-T11. Destructive lesions sternlar joint. ebrae 2-3. Lymphadenopath~. Destructive lesion r i g h t Weight loss. scapula.

3

3

M

Neck pain. Uveitis

Discospondylitis C3-C4, TII-T12.

4

3

FS

Lameness l e f t hindleg; swollen l e f t s t i f l e . Oedema l e f t hindleg. Enlarged l e f t popliteal lymph node. Weight loss, pyrexia, generalized weakness

Destructive lesion l e f t distal femur.

5

2

FS

Paraplegia, back pain. Uveitis of l e f t eye. Weight loss, pyrexia.

Discospondylitis TI3-LI.

6

3

FS

Posterior paresis and back pain. Bilateral uveitis. Weight loss, pyrexia, generalized weakness.

Not available

7

7

F

Lameness and swelling of hock. Weight loss, lethargy and vomiting.

Destructive lesion of hock

8

3

F

Malaise, pyometra.

Large uterus/kidneys

i

M = male

F = female

FS = female speyed

840 TABLE I I

BUN = blood urea nitrogen TSP = t o t a l solids protein AP = a l k a l i n e phosphatase

Laboratory Findings

Case

Haematology

Biochemistry

Urinalysis

Microbiology

i

Mature neutrophilia, Lymphopaenia (924/~I).

BUN & Creatinine elevated TSP elevated AP elevated

Fungal hyphae present

A. t e r r e u s

Mature neutrophilia. Lymphocyte count normal (2964/~I).

BUN elevated TSP elevated AP elevated

Fungal hyphae

A. t e r r e u s

Lymphocyte count normal (2952/~I).

BUN normal TSP elevated AP normal

Fungal hyphae present

A.

Mature neutrophilia. Lymphocyte count normal (2856/~I).

BUN normal TSP elevated AP elevated

Fungal hyphae

A. t e r r e u s

Mature neutrophilia. Lymphocyte count normal (1415/~I).

BUN elevated TSP elevated

Fungal hyphae present

A.

Mature neutrophilia. Lymphocyte count normal (3200/~I).

BUN normal TSP elevated AP elevated Amylase elevated

Not a v a i l a b l e .

A.'terreus

2

3

4

5

6

cultured from urine, j o i n t f l u i d carpus.

cultured from urine, lymph node aspirate, joint fluid shoulder terreus

cultured from urine and aspi r a t e C3-C, disc.

cultured from urine, aspirate from femor al lesion terreus

cultured from urine

isolated from urine.

A. terreus

Not

8

Not a v a i l a b l e

isolated from f i n e needle aspirate of hock l e s i o n , l i v e r & kidney lesions.

available

BUN elevated

No t

a v a i l a b 1 e

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TABLE I I I Necropsy Findings

Case 1

Disseminated aspergillosis. Focal necrotic lesions and fungal hyphae seen in vertebrae T3-T4, radial carpal bone, myocardium, kidney, spleen, pancreas, l i v e r , bone marrow, posterior chamber eye. A. t e r r e u s cultured from lesions. Disseminated aspergillosis. Pyogranulomatous lesions of lymph nodes, kidneys, vertebrae and scapulohumeral j o i n t . Not available for necropsy Osteomyelitis l e f t femur. Tenosynovitis l e f t s t i f l e j o i n t . Pyelonephritis l e f t kidney Osteomyelitis T13. Focal necrotic lesions and fungal hyphae in spleen, heart, kidneys and posterior chamber of eye. Focal necrotic lesions and fungal hyphae in kidney, spleen, l i v e r , lungs and myocardium. Pyogranulomatous lesions of l i v e r and kidneys. Pyelonephritis. Focal necrotic lesions and fungal hyphae in myocardium, kidney, uterine stump. Pyelonephritis.

Normal

Normal

Normal

Normal

Normal

Normal

Case

1

2

3

4

5 Day 1

Day 2

3200

3200

2400

2200

2100

1600

130

130

115

115

52

115

(mg/lO0 ml

IgG IgM Normal Normal 1500±500 150±50

22

18

48

100

56

34

None

None

A. t e r r e u s

Serum Antibody to

Ant igenaemia

Antigenaemia

None

>I00

>100

>100

>i00

>100

>100

>100

>100

>i00

>I00

>100

>100

C3 Level (units) Normal >66

82

N.T.

N.T.

N.T.

N.T.

139

CHs0 (units) Normal >52

COMPLEMENT C4 Level IgA I m m u n o - (units) Normal precipNormal 100±60 i t a t i o n >65

N.T. = Not Tested * Normal Dog Tested Concurrently

Serum Electrophoresis

HUMORAL

Summary of Immunological Data

TABLE IV

26.9 (23)*

3.4 (7.2)*

N.T.

N.T.

N.T.

1.1 (12.3)*

PHA Transforma t io n index

None

None

N.T.

N.T.

N.T.

None

N.T.

Anergic

N.T.

N.T.

N.T.

Anergic

T Cell IntraResponse dermal to Skin A. t e r r e u s Test

CELL MEDIATED

N.T.

N.T.

N.T.

1.4

C4 Allotype

IMMUNOGENETIC

W~

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Immunological Data A summary of the immunological status of five cases is presented in Table IV. Serum Protein Electrophoresis.

No abnormality was detected on serum

electrophoresis. Immunoglobulin Determinations.

Immunoglobulinlevels showed no consistent

d e f i c i t , however there were low IgA levels in cases 1 (34 mg/100 ml) and 5 (22 mg/100 ml) and a low serum IgM concentration in case 2 (52 mg/lO0 ml).

All

five cases had an elevated serum IgG concentration. Serum Antibody to A.

terreus.

in Table IV and shown in Fig. I.

Results from this analysis are summarized Serumfrom case 4 produced two precipitation

lines against the antigen, indicating antibody was produced in this animal to two antigenic components of A.

terreus.

NO precipitation was recorded with this antigen in case 1, 2, 3 or 5.

How-

ever sera from cases 2 and 3 produced precipitation lines with that from case 4, indicating the presence of free serum antigen in these animals.

Furthermore,

the spurred precipitation line in these cases indicated partial identity of this antigen to the prepared test antigen.

No precipitation was observed for

the twenty four control dogs tested.

Fig. 1.

Precipitation in agar gel Wells

I

- 4 : A.

terreus

antigen

Well A

: Canine case

Well B

: Positive control rabbit antiserum to A. terreus.

344

Serum C3 and C4 Quantitation.

Serum levels of complement C3 and C4 f o r

a l l cases were within normal range as presented in Table IV. Total Haemolytic Complement. Suitable samples f o r assessment of t o t a l haemolytic complement were a v a i l a b l e from only case i (139 units) and case 5 (82 u n i t s ) .

These values were within the normal range as presented in Table

IV. Lymphocyte Blastogenesis.

The transformation index using PHA in case i was

decreased (1.1) compared to the normal control dog (12.3).

In case 5, the

index was also below the control sample on day i (3.4; control 7.2) but was normal on day 2 (26.9; control 23). A.

terreus

There was no response to the prepared

antigen in e i t h e r case or the control dogs.

In tradermal Skin Testing.

In both dogs tested ( i and 5), reaction to the

test antigens was considered negative at 20 minutes and 24 hours post i n j e c t ion.

However, a minimal response to histamine also suggested the animals may

have been generally unresponsive at the time of t e s t i n g . Complement C4 A l l o t~ p i n g . from only 2 of the 8 dogs.

Suitable samples f or C4 a l l otyping were a v a i l a b l e Case 1 was found to have the phenotype C4-1,4 com-

pared with 14% of a random population of 92 normal dogs and 10% of a random population of 31 normal German Shepherds.

Case 2 had the phenotype C4-4 which

was present in 22% of the random population and 35% of the random German Shepherd population. DISCUSSION Opportunistic a s p e r g i l l o s i s may occur secondary to chronic d e b i l i t a t i n g disease in man, and canine nasal a s p e r g i l l o s i s has also been associated with such conditions (Dawson et a l . , 1973; Barrett et a l . , 1977). study had a previous h i s to r y of chronic i l l n e s s .

No dog in this

Long term c o r t i c o s t e r o i d

administration, a n t i b i o t i c therapy or i r r a d i a t i o n may also enhance systemic mycoses (Zimmerman, 1955).

However no case in this series received such t r e a t -

ment. Geographical l o c a l i z a t i o n of a disease caused by a ubiquitous micro-organism may be related to c l i m a t i c factors.

Most of the cases in this series were

presented during the hot, dry summer months which is in accordance with the hypothesis of Laham and Carpenter (1982) that in man these conditions may favour propogation of this pathogen.

Human disseminated a s p e r g i l l o s i s has been

reported from Texas (Laham and Carpenter, 1982) and three of the four reported canine cases are from C a l i f o r n i a (Wood et a l . , 1978; Weitkamp, 1982).

I t may

be relevant that these regions have summer conditions s i m i l a r to Western Aust-

345

ralia. The most l i k e l y explanation f o r the r e s t r i c t i o n of t h i s condition to i n d i v iduals of the German Shepherd breed is that an in h e r it e d general or s p e c i f i c immune defect exists which permits p r o l i f e r a t i o n and dissemination of A. t e r r eUS.

The disease did not manifest u n t i l the animals were at le a s t two years of age, suggesting a secondary immunological defect.

A l t e r n a t i v e l y , a primary

immunological problem may e x i s t but remain subclinical u n t i l the optimum combination of predisposing conditions occurs. In the group of dogs under study, there was no consistent humoral d e f i c i t , however, two dogs had decreased serum levels of IgA and one animal had a decrease in serum IgM.

A consistent feature in a l l cases was an elevated level

of IgG. I d e n t i f i c a t i o n of specific antibody to A. t e r r e u s by double d i f f u s i o n has been shown to be useful in providing a rapid serological diagnosis in three out of four human cases (Seligsohn et a l . , 1977; Laham and Carpenter, 1982). In t h i s series, serum antibody to A. t e r r e u s using gel d i f f u s i o n was present in only one dog, while antigenaemia was demonstrated in two other cases.

The

lack of specific antibody in the face of raised immunoglobulin G levels points e i t h e r to a f a i l u r e to produce any specific antibody or binding of a l l a n t i body producing immune complexes.

The fact that in 2 cases antigenaemia was

detected supports the l a t t e r suggestion. Budzki and Negroni (1976) postulated that complement i n h i b i t o r y factors may play a role in the pathogenesis of human A. fu~nigatus i n f e c t i o n , however in these dogs a l l serum complement l e v e l s measured f e l l within normal l i m i t s . A depression of response to PHA r e l a t i v e to normal controls, in the presence of normal lymphocyte numbers may suggest a problem in cell mediated immune function, although the opportunity was only a v a i l a b l e to test two animals.

This f i n d i n g , combined with the lack of response observed on skin t e s t -

ing, most l i k e l y r e f l e c t s the chronic and d e b i l i t a t i n g disease state present at the time of sampling rather than a primary immunological f a i l u r e .

Depressed

PHA response was reported in one other case of canine disseminated a s p e r g i l l osis (Wood et a l . , 1978) and in three cases of nasal a s p e r g i l l o s i s where depression of response to con A and PWM were also recorded (Barrett et a l . , 1977). The c l i n i c a l and immunological data suggest that a s p e c i f i c i n a b i l i t y to respond to A. t e r r e u s may e x i s t in the German Shepherd breed in Western Australia.

Whilst only l i m i t e d pedigrees were a v a i l a b l e f o r analysis, the small

genetic pool and p o p u l a r i t y of the breed in this State may enhance the presence of such a t r a i t and combined with optimal c l i m a t i c conditions may account f o r the geographical clustering of the disease.

346

I f such a defect does e x i s t , i t s genetic basis may be associated with the Major H i s t o c o m p a t i b i l i t y Complex (MHC). The role of the MHC in immunological phenomena is now well accepted.

A number of mechanisms have been postulated

to explain the association between MHC antigens and the s u s c e p t i b i l i t y to pathogens.

These include antigenic mimicry, MHC antigens acting as receptors

for organisms, the e f f e c t of the MHC on c e l l u l a r responsiveness and the presence of s p e c i f i c immune response genes (Bodmer and Bodmer, 1978). HLA linkage to the c l i n i c a l course of i n f e c t i o u s disease has been described (De Vries et a l . , 1976) and i t is therefore possible that the canine histocomp a t a b i l i t y complex (DLA) may be associated with a l t e r a t i o n of the immune system predisposing to systemic A. terreus i n f e c t i o n .

In t h i s event, polymorphic

complement C4 and DLA antigen associations may prove to be useful in understanding the pathogenetic mechanisms of such immune mediated disease. ACKNOWLEDGEMENTS The authors wish to acknowledge the co-operation of Dr B Chapman and Dr D Watson and the technical assistance of Mr A D Lines and Mr M King (School of Veterinary Studies, Murdoch U n i v e r s i t y ) .

We g r a t e f u l l y acknowledge the i n v a l -

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