Richard Malik Centre for Veterinary Education The University of Sydney

Transcription

1 Richard Malik Centre for Veterinary Education The University of Sydney 1 Pathology Update 3/8/2019

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9 9 You have to understand its environmental niche Pathology Update 3/8/2019

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36 36 Birds have adaptive immunity B cells T cells etc Pathology Update 3/8/2019

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42 Identification of the likely point source of infection in a case of avian cryptococcosis Laura Schmertmann 1,2, Kate Bodley 3, Mark Krockenberger 2, Richard Malik 4 & Wieland Meyer 1 Contact: Laura.Schmertmann@sydney.edu.au 1 Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology, Sydney Medical School Westmead Hospital, Marie Bashir Institute for Infectious Diseases and Biosecurity, The Westmead Institute for Medical Research, The University of Sydney, Sydney, NSW, Australia; 2 Faculty of Veterinary Science, The University of Sydney, Sydney, NSW, Australia; 3 Zoos Victoria, Melbourne, VIC, Australia; 4 Centre for Veterinary Education, The University of Sydney, Sydney, NSW, Australia. Introduction Cryptococcosis, an invasive fungal disease caused by members of the Cryptococcus neoformans/c. gattii species complex, affects humans and various animal species worldwide. Eucalypt hollows are a common environmental niche for C. gattii 1 and are frequently used by many Australian native bird species, including red-tailed black cockatoos (Calyptorhynchus banksii), for nesting and shelter 2. A captive red-tailed black cockatoo (Figure 1) in Melbourne Zoo was diagnosed with localised cutaneous cryptococcosis caused by C. gattii. This study aimed to identify the source of infection and assess the environmental presence of C. gattii in two aviaries inhabited by this cockatoo and numerous other birds. Results Cryptococcus spp. colonies were identified in 4/13 environmental samples, all from a single tree hollow in a dead eucalypt (Figure 2A). Environmental cryptococcal isolates (n=10) were identified as C. gattii molecular type VGI (n=9) and C. neoformans molecular type VNI (n=1). The disease isolate was identified as C. gattii molecular type VGI. MLST analysis of all C. gattii isolates identified the disease isolate as ST 159 and the environmental isolates as STs 51 (n=1), 57 (n=6) and 154 (n=2). Variation between isolates was seen only at the GPD1 and SOD1 loci, with two and three unique allele types identified respectively. STs 159 and 154 differ only by a single base-pair insertion at the GPD1 allele. Maximum likelihood phylogenetic analysis confirmed an extremely close relationship between these two sequence types (Figure 3) WM (ST 57) WM (ST 57) WM (ST 57) WM (ST 57) WM (ST 57) Figure 1: A captive red-tailed black cockatoo with a localised cutaneous cryptococcal lesion (featherless region caudoventral to the right eye). Methods Environmental samples (n=13), including pooled substrate (n=2), surface swabs (n=5) and tree hollow (Figure 2A) debris (n=6), were inoculated onto birdseed agar and incubated at 27 C. Cryptococcus spp. colonies were identified by a brown colouration (Figure 2B) and two to three colonies from each positive sample were subcultured on Sabouraud s agar at 37 C for isolation and DNA extraction. A disease isolate was available from the cockatoo. Restriction fragment length polymorphism analysis of the URA5 gene determined species and molecular type 3. Multi-locus sequence typing (MLST) analysis of seven loci (CAP59, GPD1, IGS1, PLB1, LAC1, SOD1 and URA5) using the ISHAM consensus scheme determined the sequence type (ST) 4. Phylogenetic analysis was performed using concatenated MLST sequences and the MEGA7 software package. WM (ST 159) WM (ST 154) WM (ST 154) WM (ST 57) WM (ST 51) Figure 3: Evolutionary tree of C. gattii isolates inferred by maximum likelihood phylogenetic analysis; green and red represent environmental and disease isolates respectively; scale bar represents the number of substitutions per site. Conclusions The culture and genotyping results are supportive of a single tree hollow as the likely source of infection for this cockatoo. Careful removal or decontamination of the tree may reduce the risk of further cases of cryptococcosis occurring in the population of this aviary. A Figure 2: A) An aviary inhabited by the cockatoo with a tree hollow (red arrow) that was sampled for this study and B) Birdseed agar culture of an environmental sample with Cryptococcus spp. colonies exhibiting the brown colour effect. B The absence of an identical match between the disease and environmental sequence types could be a reflection of the small sample size or of microevolutionary changes to the pathogen after contact with the host. Future work that may help to clarify this and offer further insights into the hostpathogen-environment interactions involved in cryptococcosis could include: Collection of further environmental isolates for analysis Whole genome sequencing for higher resolution of genetic variation. Acknowledgments This work was supported by the Faculty of Veterinary Science Student Support Grant Scheme at The University of Sydney. We thank Aziza Khan, Krystyna Maszewska and Veronica Ventura for laboratory assistance. Pathology Update 3/8/ References 1 Ellis, D.H. & Pfeiffer, T.J., Natural habitat of Cryptococcus neoformans var. gattii. Journal of Clinical Microbiology, (7): p ; 2 Goldingay, R.L., Characteristics of tree hollows used by Australian birds and bats. Wildlife Research, (5): p ; 3 Meyer, W., et al., Molecular typing of IberoAmerican Cryptococcus neoformans isolates. Emerging Infectious Diseases, 2003, 9(2): p ; 4 Meyer, W., et al., Consensus multi-locus sequence typing scheme for Cryptococcus neoformans and Cryptococcus gattii. Medical Mycology, 2009, 47(6): p

43 43 SIX TIMES AS COMMON IN CATS THAN DOGS Pathology Update 3/8/2019

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46 Localised cutaneous disease Localised nasal cavity disease Pathology Update 3/8/

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49 NASOPHARYNGEAL CRYPTOCOCCOSIS Canada crypto 3/8/

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52 Pathology Update 3/8/

53 LOCALISED NASAL CAVITY DISEASE WITH CONTIGUOUS SPREAD TO THE NASAL PLANUM OR NASAL BRIDGE BEFORE AFTER THERAPY 3/8/2019 Pathology Update 53

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73 BEFORE AND AFTER THERAPY Canada crypto 3/8/

74 JET-SETTING KOALAS Canada crypto 3/8/

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77 Humans have poorly developed nasal cavity (reductive evolution) Pathology Update 3/8/

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81 VIRULENCE STUDIES Canada crypto 3/8/

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84 1. First step is COLONISATION This can be demonstrated by collecting deep nasal washings (and sometimes even superficial swabs) and plating on Staib s bird seed agar the only colonies that develop brown colour effect are Cryptococcus spp C. gattii VGI tends to have highly mucoid colonies Note that there are many fungi in the normal mycobiome of cats and dogs Canada crypto 3/8/

85 1. First step is COLONISATION 2. Something facilitates INVASION of the epithelium FHV-1? Canada crypto 3/8/

86 1. First step is COLONISATION 2. Something then facilitates INVASION of the epithelium FHV-1? 3. This gives rise to LOCALISED INFECTION Nasal turbinates (mouse) Canada crypto 3/8/

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88 COLONISATION Canada crypto 3/8/

89 Early INVASION of the epithelium Mycology Masterclass 3/8/

90 LOCALISED INFECTION Mycology Masterclass 3/8/

91 The HOST responds by non-specific cellular response (neutrophils, macrophages) which is LATER organized by SPECIFIC CMI (cell mediated immunity) Mycology Masterclass 3/8/

92 MANY ANIMALS WILL HAVE SUBCLINICAL DISEASE DUE TO AN EFFECTIVE IMMUNE- MEDIATED RESPONSE Use antigen testing (IMMY lateral flow or latex cryptococcal agglutination [LCAT]) to demonstrate infection (subclinical or clinical) Clinical disease gives rise to signs and symptoms Canada crypto 3/8/

93 LCAT/IMMY Negative Positive (low) Positive (higher) Colonization Subclinical Disease Clinical disease Mycology Masterclass 3/8/

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