Disseminated Aspergillosis in a Whooper Swan (Cygnus Cygnus)161-174).pdf · 2011-05-10 ·...

Egypt. J. Comp. Path. & Clinic. Path. Vol. 21 No. 2 (April) 2008; 161- 174

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Disseminated Aspergillosis in a Whooper Swan (Cygnus Cygnus)

By

Abou-Rawash, A. A.1*; Yanai2 T.; Kano3 R.; Murakami2 M.; Masegi2

T.; Fukushi2 H.; Nagamine3 T. and Hasegawa4 A. 1 *Path. Dept., Fac. of Vet. Med., Alex. Univ., Damanhor Br., Elbostan, Egypt.

2 Vet. path.Dept, Gifu Univ., 1-1 Yanagido, Gifu, Japan. 3Nagamine Animal Clinic, Maehara, Gusigawa, Okinawa, Japan. 4Pathobiol.Dept.,Nihon Univ., Sch. Vet. Med. Kameino, Fujisawa,

Kanagawa, Japan.

SUMMARY A Whooper Swan which usually migrates from Siberia to the north of

Japan in the winter season was found in Kume Island at the south of Japan in a state of pronounced illness. Blood and serum analysis revealed hypoalbuminaemia elevated serum level of uric acid (UA) and creatinin phosphokinase (CPK). The bird succumbed after unsuccessful attempts for treatment. At necropsy, the bird had multiple focal and coalescent caseated granulomatous nodules of whitish greenish color on the thoracic and abdominal air sacs, the lungs, and the serosal surfaces of the spleen, liver and kidneys. At the inner side of the air sacs hyphal growth with dark greenish color were scattered on most of the surface. Histopathologically, the present case had a sever degree of chronic disseminated aspergillosis. The granulomas had a central necrotic areas consisted of necrotic cell debris and hyphae with the microscopical features of Aspergillus. A. fimigatus was identified in tissues by PAS, GMS, and immunohistochemically. PCR was very successful to identify the causative fungus like other infectious agent.

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Prof. Dr. Salah Deeb Deeb Professor of Pathology, Fac. Vet. Med., Beni Suef University

Prof. Dr. Eman Bakr Professor of Pathology, Fac. Vet. Med., Cairo University

INTRODUCTION

spergillosis, as a term was firstly applied to respiratory

disease caused by fungi of aspergillus spp. as early as 1842 by Rayer and Montagne (Friend and

Franson, 1999) is now a world wide distributed. Aspergillosis is not only a disease of birds but also it has been reported in nearly all species of the kingdom animalia (Atkinss-on and Brojer, 1999;

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Friend and Franson, 1999 and Oglesbee, (1997). The disease is caused by fungal species of genus Aspergillus (A). Although A. falv-us, A. niger, A. nidulans and A. terreus were all isolated from cases of Aspergillosis, A. fumigatus rem-ains the most frequently isolated species, (95%) (Heidenreich, 1997; Latgé, 1999 and Stone and Okoniewski, 2001). A. fumigatus was first isolated from lung of bustard (Otis tardaga) in 1863 (Chute and Richard, 1991). It has been proved that Aspergillus fumigatus is thermophilic species with growth occurring at temperat-ure as high as 55°C and growth ma-intained at temperature up to 70 °C (Latgé, 1999). The disease in birds is usually of two forms acute and chronic. The acute form as a result of inhaling large number of spores is characterized by diffuse coloniz-ation and infiltration of the lung with milliary granulomas. The chr-onic form which usually follow a stressful condition (as malnutrition, starvation, immunosuppression, lo-ng antibiotic therapy) is the most common form in wild birds which could be either focal or generalized (Atkinsson and Brojer, 1999; Fri-end and Franson, 1999 and Ogl-esbee, 1997). In this study we do not only report the chronic gener-alized (disseminated) A. fumigatus infection in a breed of wild birds which had not been documented to contract the disease before, the

Whooper Swan, but we also descr-ibed a successful molecular ident-ification of the DNA extracts of A. fumigatus by PCR technique.

MATERIALS AND METHODS

Whooper Swan which usua-lly migrates from Siberia to

the north of Japan in the winter season was found in Kume Island at the south of Japan in a state of pronounced illness with dropped wings, knocked head, and marked dehydration, and emaciation. Blood and serum analysis revealed hypo-albuminemia and elevated serum level of uric acid (UA) and crea-tinin phosphokinase (CPK) (Serum albumin was 1.1 g/dl, UA was 13.7 mg/ dl; (reference range 2.9- 3 mg/dl), CPK was 87.5 U/liter). Few organisms of Trychomonas spp. were isolated from the feces. The bird was treated with the rec-ommended doses of Bytril, Ringers and Glucose and then supplied with liquid diets. The bird temporarily responded to the treatment and gained weight. Twenty days later marked health deterioration with loss of appetite, and marked dys-penia were observed. Ten days later, the bird succumbed after sev-eral unsuccessful attempts for trea-tment with the recommended doses of Bytril (enrofloxacin), 4 ml S.c.; Fungizon (amphotracin B) 2 ml int-ratracheal and complementary vit-amins, mineral, nutritive and fluid therapy. Fuloconazol 3 ml S.c. was

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also used as a treatment trial. Blood and serum analysis two days before death revealed mild anemia hypoa-lbuminaemia and marked increases in serum UA and lactic dehydroge-nase LDH. (26% hemtocret (Ht), 1 g/dl serum albumin, 199 mg/dl UA and 622 U/liter LDH ).

Post mortem examination was conducted and tissue specimens fr-om organs were collected, fixed in 10% formalin and processed for paraffin embedding and sectioning at 4 μ thickness for routine histopa-thological technique. The sections were then mounted and stained wi-th hematoxylin and eosin (H&E), periodic acid Schiff (PAS), Grocott methamine silver (GMS). Immunohistochemistry:

Other histological sections were immunohistochemically stai-ned with immuno-enzyme polymer method using Histopfine simple stain MAX-PO Kit (Nichuirei Corp., Tokyo, Japan). The sections were treated with 0.1% trypsin and endogenous peroxidase was block-ed by methanol and 3% H2O2 prior to immunostaining. Mouse anti-Aspergillus fumigatus wall fraction monoclonal antibodies (Mab-WF-AF-1, DAKO, Carpinteria, USA), mouse anti-Rhizopus arrhizus wat-er-soluble somatic antigens mono-clonal antibody (Mab-WSSA-RA-1, DAKO, Carpinteria, USA), and Rabbit polyclonal antibodies aga-

inst Candida albicans (1750-5507 Biogenesis, Poole, Dorset, Engla-nd) were used as primary antibod-ies. Simultaneously positive (tiss-ues infected with the fungi in con-cern) as well as negative control (by using non immune respective sera instead of the primary anti-bodies) were stained. The sections were then lightly counterstained with hematoxylin, mounted in aqu-eous mounting media and exami-ned by light microscopy.

For fungal isolation samples aseptically collected from the org-ans showing gross lesions (the air sacs and the lungs) were cultured aerobically on Sabouroud’s dextro-se agar at 25 °C for 1 week. Isola-ted fungus was identified according to gross morphological criteria as well as microscopically (Haines, 1995; Chung and Bennett 1992 and Samson and Van Reenen, 1988).

Polymerases Chain Reaction (PCR):

Further identification using polymerase chain reaction (PCR) was also performed on the isolated fungus using the 28S ribosomal DNA for confirmation of the mor-phological identification. The 28S primers used were 5'-GCA TAT CAA TAA GCG GAG GAA AAG-3' and 5'-GGT CCG TGT TTC AAG ACG-3' (S-1 forward primer


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and S-2 reverse primer respectiv-ely). With these primers, a 600 bp fragment of the sequences of 28S rRNA gen was expected to be amplified. The genomic DNAs were prepared by the method reported previously by Kano et al., (2002)15. The DNA (100 ng) samples were amplified by PCR in a reaction mixture (30 ml) contain-ing 10mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin, 200 mM each of deoxy-nucleoside triphosphate, 1.0 unit of Taq polymerase and 0.5 mg of a pair of primers. The PCR ampli-fication was carried out for 35 cyc-les consisting of template denatu-ration (1 min, at 94oC), primer an-nealing (2 min, at 55oC) and pol-ymerization (3 min, at 72oC). The PCR products from the sample was sequenced by the dideoxy chain termination method (Big dye Term-inator Cycle Sequencing Ready Reaction Kit; Applied Biosystems, Foster City, CA, U.S.A.) with an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, U.S.A.).

RESULTS

t necropsy, the bird was ma-rkedly emaciated and had

multiple focal and coalescent caseated granulomatous nodules of whitish greenish color on the thoracic and abdominal air sacs, as well as on the serosal surfaces of spleen, liver and kidneys. At the

inner side of air sacs hyphal growth with dark greenish color were scattered on most of the surface. The lungs were severely affected and the granuloma were not rest-ricted only to the surface but white grayish caseous nodules of 1-3 cm in diameter were scattered through-out the entire parenchyma (Fig. 1 A & B). Histopathologically the thora-cic and abdominal air sacs walls were markedly thickened by accu-mulation of closely packed necrotic cell debris and inflammatory cells. Serofibrinous exudate enmeshed with numerous fungal hyphae were coating the inner side of the thi-ckened air sacs wall (Fig. 1C )

The lung showed presence of

multiple granulomas formed of he-avy cellular infiltration of neutro-philes, lymphocytes, macrophages, and occasional multinucleated gia-nt cells (Fig. 1-D). The granulomas had a central necrotic areas consist-ed of necrotic cell debris and hyph-ae with the microscopical features of Aspergillus spp (Fig. 2A). Some granulomas were coalesced formi-ng diffuse granulomatus reaction with multiple necrotic areas conta-ining the fungal organism. Sero-fibrinous exudate contain moderate number of inflammatory cells exis-ted in the lung tissue among the granulomatous nodules. The liver showed multiple small-sized focal

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necrotic areas, most of which were located at the periportal region. Periportal infiltration of plasma cells lymphocytes, and macropha-ges were frequently observed. The fungal hyphae were demonstrated in the affected tissues of the lung, air sacs, and serosal covering of the kidneys, spleen, intestine and gizz-ard. GMS stain and PAS reaction revealed presence of GMS and PAS positive fungal hayphae with-in the granulomas in all affected organs. (Fig. 2B, 2C) Immunohist-ochemistry for Aspergillus fumig-atus revealed strong positive react-ion in all granulomas of the lungs and air sacs and serosal covering of visceral organs (Fig. 2-D). Weak positive reaction against Candid albicans was also detected in some of the air sacs lesions. No positive reaction was detected against Rh-izomucur in any organ. No specific reaction was detected in heart muscle or nervous tissue. Although the serosal covering of the intestine showed granulomatous reaction po-sitive for Aspergillus fumigatus, so-me of the nodules with unidentified

helminthes parasite were negative for the three fungi examined.

The results of fungal isolation revealed presence of fungus having the diagnostic morphological crite-ria of Aspergillus fumigatus on Sabaroud’s dextrose agar solid me-dia as well as microscopically. The results of PCR amplification of the DNA extract of the isolated fungus with 28S ribosomal DNA primers yielded nucleotide fragments of about 600-bp. (Fig. 3 ).

Homology relationships amo-

ng the 28S ribosomal DNA of reference strains of Aspergillus species and that of the clinical isolate of the present study were examined by FASTA data base analysis in DNA data bank of Ja-pan (DDBJ). The sequence of the amplicone consistent with the seq-uence of 28S ribosomal DNA sequences from fungal species. It revealed 100% homology with Aspergillus fumigatus (DDBJ accession no. AJ438344).


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Figure 1. (A) Showing growth of A. fumigatus on abdominal air sac. (B)

Thoracic air sac showing extensive fungal growth. (C) Dense fungal growth overlying the confluent granulomatous reaction in air sac H&E X 200. (D) Lung showing mix of cellular infiltration notice epthelioid and giant cells (arrow)H & E X 400.


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Figure 2. (A) Air sac showing confluent granulomatous reaction with many

fungal hyphae (arrows) and conidial heads H&E X 200. (B) PAS positive hyphae and conidial heads on the surface of air sac PAS X 200. (C) Lung showing GMS positive hyphae (black) inside granulomatous foci, incent showing hyphae with conidial head GMS X 100. (D) Strongly immune positive hyphae of A. fumigatous within granulomatous foci in the lung. immunoperoxidase using anti- A. fumigatus wall fraction monoclonal antibodies. X 200.


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Fig. (3): Showing 600 bp of nucleotide DNA fragment extract of isolated

fungus.

DISCUSSION

nterestingly the earlier inv-estigator believed that fungi fou-

nd in lesions of avian species were growing saprophytically (Austw-ick, 1965). Recently aspergillosis has been reported in most of the domestic and wild birds. It had been reported that immunocomp-etent hosts usually effectively eliminate the conidia of A. fumiga-tus (Schaffner, 1994). The birds usually acquire aspergillus spp. infection through inhalation of a huge number of conidia especially when they are immunocompromis-ed (Oglesbee, 1997 and Reidg, 1993). Although Chute and Rich-ard, (1991), mentioned occurrence of molds likely belong to Asper-gillus spp. in wild birds including

swans as early as 1800, Aspergill-osis in swan is very poorly documented. In a survey study of lead concentration and associated mortalities in Tundra Swan, Law-rence et al. (1999), briefly ment-ioned gross appearance of one case with lung aspergillosis. To the best of our knowledge no documented report were published demonstra-ting the causative fungal species using a nearly established, undoub-ted molecular biology techniques. In the present study a Whoo-per swan had a disseminated aperg-illosis caused by fumigatus species, identification was based on detect-ion of the hyphae within grnulom-atous lesions by H&E, PAS and GMS stain and immunohisto-

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chemistry as well as macroscopic and microscopic identification of the isolated fungus as A. fumigat-ous on Sabaroud agar. Histopathol-ogically the present case had a sever degree of chronic dissemin-ated aspergillosis in the air sacs, lungs and serosal covering of the liver, kidneys, spleen, and intestine. Similar findings were reported in aspergillosis in chickens, water fo-wls, turkeys, ostrich and wild birds (Chute and Richard, 1991 and Friend and Franson, 1999) GMS stain, PAS reaction and immuno-histochemistry indicated no evide-nce of vascular invasion or hem-atological spread as no fungus was detected in blood vessels or within any organ other than the respiratory system. Spread of the infection from the lung to the serosal cov-ering of the liver, spleen, intestine, and kidneys might be direct extens-ion of the lesions in the lungs and air sacs. This was supported by the absence of any fungus in the pare-nchyma of other organ. The focal necrotic areas in the liver might be attributed to the effect of absorbed soluble toxins and metabolic enzy-mes presumably generated by fun-gal growth in the air sacs and lun-gs, as it had been proved that A. fumigatus produce several array of toxins and putative virulence facto-rs enzymes (Sutton et al., 1994). The perivascular mononuclear cell infiltration in the portal areas could be direct reaction to the absorbed

fungal soluble antigens. Presence of weak positive reaction against Candida albicans antibodies as de-monstrated by immunohistoche-mistry could be explained as seco-ndary infections as a result of imm-unosuppression caused by Asperg-illus spp. Absences of hematolo-gical spread in this report together with absence of previous reports about aspergillosis in Whooper sw-an may indicate the presence of some sort of innate resistance of this species swan to A. fumigatus infection. In a cross breeding study Heidenreich, (1997) reported inna-te resistance to Aspergillosis in gy-rfalcon. In addition, molecular ide-ntification by PCR on the DNA ex-tracts of the isolated fungus revea-led A. fumigatus as a final diagno-sis, which confirmed the histopath-ological and morphological diagn-osis. PCR was also very successful to identify the causative fungus like other infectious agent. Several targ-ets have been used for the identifi-cation of fungi, including mitocho-ndrial DNA, chitin synthase, P450, heat shock protein, actin, and rRNA gene. (The DNA that encod-es ribosomal RNA is referred as rRNA gene (Chryssanthou et al., 1994; Ferrer et al., 2001; Jordan, 1994; Kan, 1993 and Miyakawa et al., 1993). Among these, rRNA gene is most suitable because of its conservative nature. The genes en-coding rRNA contain a small sub-unit (SSU), 18S rRNA and 5.8S


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rRNA, and a large subunit (LSU), 28S rRNA. Internal transcribed sp-acer (ITS) regions I and II are more variable than the rest of the riboso-mal gene subunits and are found between SSU rRNA-5.8S rRNA and 5.8S rRNA-LSU rRNA, respe-ctively. Because LSU and SSU are the most conserved regions, identi-fication of their nucleotide polymo-rphism may lead to the different-tiation between fungal species (Bialek et al., 2000; Hinrikson et al., 2005 and Ninet et al., 2003). rRNA gene is considered to be the most conserved region of fungal genome and has been used by se-veral workers to identify fungi for diagnosis of diseases, to explore biologic diversity, and to identify plant pathogens (Hennequin et al., 1999; Karen et al., 1997 and Kur-tzman and Robnett, 1997). Howe-ver, in the present study, we succ-essfully used a sequence of a fragment of this most conserved LSU region to identify the fungal pathogen through available databa-ses up to the species level. Kumar and Shukla, (2005 and 2006)

reported that LSU has more seque-nce variation than SSU and can be used for strain level differentiation and identification of fungi. The collective results of the present report clearly demonstrated the pathological changes and the causality relation of death of this Whooper swan and A. fumigatous.

We should also mention that, this is the first record of aspergillosis as a result of A. fumigatus infection in this breed of swan and to the best of our knowledge in this species of migratory wild birds. Further inve-stigation is required to evaluate innate resistance of this species to aspergillosis. Acknowledgement:

his study was partially supported by 21st Century -of-

Excellency Program .

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