Ventral dermatitis in rowi (Apteryx rowi) due to cutaneous larval migrans B.D. Gartrell a,*, L. Argilla b, S. Finlayson a,b, K. Gedye a, A.K. Gonzalez Argandona a,b, I. Graham c, L. Howe a, S. Hunter a, B. Lenting b, T. Makan d, K. McInnes d, S. Michael a,b, K.J. Morgan a, I. Scott a, D. Sijbranda a,b, N. van Zyl a, J.M. Ward a a Wildbase, Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North 4410, New Zealand b Wellington Zoo, 200 Daniell Street, Newtown, Wellington 6021, New Zealand c Department of Conservation, Franz Josef Office, State Highway 6, Franz Josef Glacier, 7856, New Zealand d Science and Capability Group, Department of Conservation, National Office, 18-32 Manners Street, Wellington 6011, New Zealand A R T I C L E I N F O Article history: Received 15 September 2014 Revised 31 October 2014 Accepted 6 November 2014 Keywords: Apterygiformes Aspergillosis Cutaneous nematodiasis Kiwi Operation nest egg Trichostrongylus A B S T R A C T The rowi is a critically endangered species of kiwi. Young birds on a crèche island showed loss of feath- ers from the ventral abdomen and a scurfy dermatitis of the abdominal skin and vent margin. Histology of skin biopsies identified cutaneous larval migrans, which was shown by molecular sequencing to be possibly from a species of Trichostrongylus as a cause of ventral dermatitis and occasional ulcerative vent dermatitis. The predisposing factors that led to this disease are suspected to be the novel exposure of the rowi to parasites from seabirds or marine mammals due to the island crèche and the limited man- agement of roost boxes. This is the first instance of cutaneous larval migrans to be recorded in birds. Severe and fatal complications of the investigation resulted in the death of eight birds of aspergillosis and pul- monary complications associated with the use of bark as a substrate in hospital. Another bird died of renal failure during the period of hospitalisation despite oral and intravenous fluid therapy. The initiat- ing cause of the renal failure was not determined. These complications have the potential to undermine the working relationship between wildlife veterinarians and conservation managers. This case high- lights that intensive conservation management can result in increased opportunities for novel routes of cross-species pathogen transmission. © 2014 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). 1. Introduction Rowi (Apteryx rowi), also known as Okarito brown kiwi were only recognised as a unique species of kiwi (Apteryx spp) in 2003 (Burbidge et al., 2003; Shepherd and Lambert, 2008). The Depart- ment of Conservation in New Zealand classifies the population as ‘threatened: nationally critical’ (Robertson et al., 2013), and by the IUCN Red List as endangered (BirdLife International, 2014). The total population was estimated at 375 birds in 2011. The geographic dis- tribution of the wild population is confined to a single protected area of forest of approximately 11,000 hectares. This area is located northwest of Franz Josef/Waiau village on the west coast of the South Island of New Zealand. The rowi population is intensively managed by the Depart- ment of Conservation (DOC). One component of the conservation management of the species is an Operation Nest Egg programme based on the principle that the highest mortality of kiwi due to mustelid predators (especially stoats Mustela erminea), occurs during the birds’ early growth period. Eggs are removed from the burrows of wild birds, artificially incubated and hatched, and the young chicks are reared in predator free crèches. The young chicks are initially reared in indoor brooders, then placed in outside pens, and subsequently moved to a predator-free offshore island (crèche island) where they are largely independent and only monitored intermittently. Finally, they are returned to the wild population when sufficiently grown to reduce predation mortality (mean 361 days of age ± SD 106 days) (Colbourne et al., 2005). In July 2013, a Department of Conservation ranger reported that on physical checks of the 30 young birds on the crèche island, 15 birds were showing loss of feathers from the ventral abdomen and around the vent and a crusty dermatitis of the abdominal skin and Note: Nucleotide sequence data reported in this paper are available in the GenBank™ databases under the accession number KM434192. * Corresponding author. Wildbase, Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North 4410, New Zealand. Tel.: +64 6 356 9099 ext 85203; fax: +64 6 350 5714. E-mail address: B.Gartrell@massey.ac.nz (B.D. Gartrell). http://dx.doi.org/10.1016/j.ijppaw.2014.11.001 2213-2244/© 2014 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). International Journal for Parasitology: Parasites and Wildlife 4 (2015) 1–10 Contents lists available at ScienceDirect International Journal for Parasitology: Parasites and Wildlife journal homepage: www.elsevier.com/ locate / i jppaw mailto:B.Gartrell@massey.ac.nz http://www.sciencedirect.com/science/journal/ http://www.elsevier.com/locate/IJPPAW http://crossmark.crossref.org/dialog/?doi=10.1016/j.ijppaw.2014.11.001&domain=pdf vent margin. One bird had an ulcerative dermatitis of the vent margin. The ranger reported that a mild version of this condition had been seen in three birds out of 30 in 2012, but was not further investigated and had spontaneously resolved. No similar condi- tion had previously been seen in rowi of any other age group nor reported in other kiwi species. In 2013, one bird with ulcerative dermatitis of the vent margin was removed from the crèche island for treatment at the same time as a bird that showed no signs of dermatitis but had sustained a traumatic leg injury and capture myopathy. The birds were admit- ted for diagnostic work up and treatment to The Nest Te Kōhanga veterinary hospital at Wellington Zoo. DOC rangers carried out a survey of all juvenile kiwi (18 birds) on the crèche island and found 14 more rowi with signs of dermatitis. After consultation with vet- erinarians at Wellington Zoo, the Wildbase Hospital at Massey University and the DOC wildlife veterinarian, these birds were cap- tured for diagnostic investigation and treatment. This paper describes the collaborative investigation of the ventral dermatitis of juvenile rowi and the fatal complications associated with the hospitalisation of the birds. 2. Materials and methods As this was a veterinary diagnostic investigation, animal ethics approvals were not required. The handling, transport, diagnosis and management of the birds were approved by the Department of Conservation. Birds were captured by hand from roost boxes and natural burrows on the island and transported by boat, car and ferry to The Nest Te Kōhanga veterinary hospital at Wellington Zoo. Seven birds were transported to the Wildbase Hospital at Massey University and the remaining seven birds were kept at The Nest Te Kōhanga, Wel- lington Zoo. The Nest Te Kōhanga already had two rowi hospitalised, one with an injury and one with dermatitis, so nine birds in total were held in this hospital. Both institutions followed an agreed di- agnostic plan for the investigation of the dermatitis; however, there were differences in the husbandry and treatment of the birds at the different veterinary hospitals, that were largely due to different stan- dard operating protocols for patient care. Birds kept at Wildbase were housed in an indoor room with rubber matting covered by towels as substrate. Multiple roosting boxes were provided and food was offered in a communal tray. The birds were fed a captive kiwi diet consisting primarily of minced cow heart, vegetables, and a mineral supplement. Initially all the birds were housed together. Body weights were monitored daily. When three birds failed to maintain or gain weight, these birds were housed singly in stainless steel cages with rubber mat flooring covered by towels and with a roost box provided. These birds were hand-fed small meat patties of the kiwi captive diet twice daily until weights were increasing again. Initially, the two rowi kept at the Nest Te Kōhanga (one with trauma and the other with dermatitis) were fed and monitored sim- ilarly to those at Wildbase. However, when the remaining birds arrived, all the birds kept at the Nest Te Kōhanga were housed in semi-outdoor enclosures, on a natural substrate comprised of shred- ded Eucalyptus bark. The birds were housed in one group of three and one group of four initially and each individual had a roost box in which the natural substrate was covered by towels. Later, the birds were housed separately and all the natural substrate was removed and replaced with rubber matting covered by towels as substrate. Birds that were unwell clinically were housed indoors individual- ly in intensive care units with soft bedding covered by towels. Given the unique presentation of the dermatitis, a broad diag- nostic plan was implemented. Where a variation in the number of birds tested occurred this is noted. The diagnostic tests used included: 2.1. Physical examination Full physical examinations were carried out on all birds (15/15 birds). 2.2. Faecal parasitology Faecal floats using zinc sulphate as the concentrating fluid were examined at one laboratory by microscopy for evidence of gastro- intestinal parasitism (15/15 birds). The birds at The Nest Te Kōhanga hospital were screened for Cryptosporidium spp. by acid-fast stain- ing of faecal smears (8/15 birds). 2.3. Skin cytology Cytology of affected vent margin was carried out using skin scrapes of the affected areas. Samples obtained in this manner were examined under oil for parasites and tissue smears were stained with modified Wright’s stain (Diff-Quik) and assessed microscop- ically (14/15 birds). One bird died before ante-mortem skin scraping was performed. 2.4. Haematology and plasma biochemistry Blood sampling was carried out for haematology and biochem- istry in all birds 1–3 days after admission to hospital. The haematology was carried out by avian veterinarians at both hos- pitals using the same manual method of estimating white cell counts from blood smears that were stained with modified Wright’s stain (Diff-Quik). The estimated white cell counts were performed on the blood smears following the method described in Fudge (2000). Quality control was achieved by one author (BG) reviewing all haematology slides. The parameters examined included packed cell volume, total plasma solids (g/L), estimated total white cell count, and the relative ratios and absolute counts for the leucocyte types identified in the peripheral blood smears, including heterophils, lym- phocytes, and monocytes. The plasma biochemistry panel included the following analytes: aspartate aminotransferase (AST); bile acids (BA); creatine kinase (CK); uric acid (UA); Glucose (Glu); calcium (Ca), phosphorus (P), sodium (Na), potassium (K), total plasma protein (TP), albumen (Alb) and total globulins (Glob) (15/15 birds). These plasma analytes were measured by the same point-of-care biochemical analyser (VetScan, Abaxis, Union City, CA, USA) at both hospitals. 2.5. Radiography Full body radiographs using digital radiography were taken in two orthogonal views under general anaesthesia that was induced and maintained with inhalational isoflurane in oxygen (12/15 birds). Three birds died before radiographs could be carried out. 2.6. Enteric bacteriology Cloacal swabs were taken from all the birds held at the Wildbase Hospital for aerobic bacterial cultures for the presence of Salmo- nella spp and Yersinia spp by a commercial veterinary diagnostic laboratory (7/15 birds). Due to laboratory error there are no results for cloacal microbiology from the birds held at The Nest Te Kōhanga. 2.7. Skin biopsies Biopsies of the affected skin and vent margins were taken using 5 mm biopsy punches under general inhalation anaesthesia with isoflurane (8/15 birds). The remaining birds either died before sam- pling could occur or had shown significant improvement in their 2 B.D. Gartrell et al./International Journal for Parasitology: Parasites and Wildlife 4 (2015) 1–10 dermatitis following anthelmintic treatment prior to biopsy. The skin biopsies were submitted for histological examination (8/8 samples), and for fungal and aerobic bacterial culture (6/8 samples) where sample volume allowed. Three skin biopsies were dissolved over- night in pepsin/HCl and the digested material examined with a dissecting microscope for the presence of larvae. Three mildly af- fected rowi that had not been previously sampled had skin biopsies four days after anthelmintic treatment for histological examina- tion only. 2.8. Molecular diagnostics 2.8.1. PCR testing of skin scrapings for herpesvirus, poxvirus and Chlamydia Skin scrapings of the lesions were examined by specific PCR testing for the presence of herpesvirus, poxvirus and Chlamydia. The methodology for these assays has been previously described; her- pesvirus (Gartrell et al., 2009); avipoxvirus (Ha et al., 2013); and chlamydia (Gartrell et al., 2013) (10/15 birds). Not all birds at The Nest Te Kōhanga were sampled because of a miscommunication. 2.8.2. PCR testing of skin biopsies for nematode sequences Tissue from five skin biopsies that were positive by histology for the presence of nematode larvae in the dermis were assessed by molecular techniques for nematode gene sequences. DNA was ex- tracted from 10 μm sections of the five skin biopsy samples that were prepared for histological examination. The sections that were used for molecular analysis were from a sandwich cut, between two slides that showed nematodes histologically. DNA was extracted using a Qiagen DNeasy kit, following the manufacturer’s instruc- tions (Qiagen, CA, USA). PCR was performed to amplify the second internal transcribed spacer region (ITS-2) of the ribosomal (rDNA) region of the nematodes. Amplification of the nematode DNA was performed using the NC1-NC2 primer constructs (Gasser et al., 1993). NC1: 5′-ACGTCTGGTTCAGGGTT-3′ NC2: 5′-TTAGTTTCTTTTCCTCCGCT-3′ For PCR amplification the conditions were as follows; 1 U Plat- inum Taq (Invitrogen, CA, USA), 1 X PCR buffer (200 mM Tris–HCl pH 8.4, 500 mM KCl), 1.5 mM MgCl2, 200 μM dNTPs each, 1 μM of each primer, 50 μg of template DNA in a total reaction volume of 25 μL. The reactions were carried out under the following condi- tions in a SensoQuest labcycler (Germany); 94 °C, 5 min, 94 °C, 30 s, 55 °C, 30 s, 72 °C, 30 s and for 40 cycles, 72 °C, 10 min. As positive controls for PCR, DNA previously extracted from a Haemonchus contortus nematode, and a clone of the H. contortus ITS-2 region were used. A negative PCR control of water was also utilised. An aliquot of 10 μL of the PCR product was separated on a 1% agarose gel that contained 0.2 mg·mL−1 of ethidium bromide for staining. Gels were run for 1 hour at a 100 V and were visualised with a UV transilluminator. 2.8.3. Sequencing of rDNA Positive samples were then identified and the remaining 15 μL of the PCR product was cleaned using 70% ethanol and resus- pended in elution buffer (10 mM Tris HCl, pH 8.0). The clean PCR product was sent to Massey Genome Service for Sanger sequenc- ing in both directions. Paired sequence data were aligned using Geneious v 6.6.1 (http://www.geneious.com/). BLAST (Basic Local Alignment Search Tool (Altschul et al., 1990)) was used to compare the resulting sequences with published sequence data in NCBI (Na- tional Center for Biotechnology Information). PCR reactions and sequencing of the rowi samples were performed twice to confirm results. 2.8.4. Phylogenetic analysis A total of 18 nematode species with published ITS 2 regions were selected for phylogenetic comparison to the material extracted from the rowi samples (hereafter called the rowi consensus samples). We included Caenorhabditis elegans as an outlier and model system. All sequence data were obtained from NCBI and when necessary trimmed to only the ITS 2 region using Geneious v 6.6.1. Utilising the tree building function of Geneious v 6.6.1, a distance matrix was constructed using the algorithm of Tamura and Nei (1993), and tree construction was performed using the Neighbor-Joining method (Saitou and Nei, 1987). 2.9. Post mortem examination Birds that died (n = 10) during the investigation were sub- jected to a post mortem examination using standard veterinary protocols, and samples were taken for histological examination, and microbiological fungal and aerobic bacterial cultures. 2.10. Statistics Multivariate GLM (SPSS v21) was used to assess any differ- ences between the two hospitals for a range of quantitative measures including: bodyweights; haematology (packed cell volume, total plasma solids, estimated total white cell count, absolute counts for heterophils, lymphocytes, and monocytes); plasma biochemistry test results (plasma concentrations of AST, CK, UA, glucose, calcium, phos- phorus, albumin, globulin, sodium and potassium); and faecal float results (coccidia, capillaria and strongyle egg counts). The overall significance of the model (Intercept*Hospital) was assessed using Pillai’s trace. Kolmogorov–Smirnov and Shapiro–Wilk tests were used to examine the data for normality. 3. Results 3.1. Physical examination Physical examination of the birds on admission showed a range of clinical abnormalities in the ventral abdominal and peri-cloacal skin (Fig. 1). All birds (15/15) showed alopecia and broken feather stubs in this area. Seven of fifteen birds showed evidence of mild dermatitis as characterised by hyperkeratotic scurfy skin. In three more severe cases there were small ulcers and fissures on the vent margin with varying degrees of exudation and matting of feathers to the vent. 3.2. Faecal parasitology Faecal floats for parasitology detected a range of gastrointesti- nal parasites. Coccidial oocysts were present in 12/15 birds with a mean oocyst count/g of faeces of 27,197 (s.e. ± 12,364). Capillaria spp. nematode eggs were present in 7/15 birds with a mean count of 317 eggs per gram of faeces (s.e. ± 290), and strongyle-type nem- atode eggs were present in 6/15 birds with a mean count of 33 eggs per gram of faeces (s.e. ± 18.5). There was a significant difference (F = 5.309, df = 3,11, p = 0.017) in the quantitative parasite egg counts in rowi faeces from the two hospitals for coccidia, strongyle and cap- illaria type eggs (Table 1). All 8 birds tested for Cryptosporidia were negative. 3.3. Skin cytology Cytology of the skin surface was assessed in 14 birds. In 8/14 birds the cytology showed only normal epithelial squamous cells with free bacteria and no evidence of inflammation. In the remain- ing 6 birds there was evidence of acute dermal inflammation that 3B.D. Gartrell et al./International Journal for Parasitology: Parasites and Wildlife 4 (2015) 1–10 was characterised by heterophils, eosinophils and abundant bac- terial rods and cocci. 3.4. Haematology and plasma biochemistry There was a significant difference (F = 263.691, df = 7,5, p < 0.001) in the haematology of the rowi kept in the two different veteri- nary hospitals (Table 2) taken in the first three days after hospitalisation, in particular the estimated white cell count (both with (p = 0.001) and without (p < 0.001) correction for PCV), and the absolute numbers of heterophils (p = 0.001) and monocytes (p = 0.015) were significantly higher in birds held at The Nest Te Kōhanga, Wellington Zoo. While specific reference ranges for rowi are not available for haematology parameters, when we extrapo- lated from other kiwi, all the birds at The Nest Te Kōhanga were showing a marked leukocytosis, with an absolute heterophilia, lym- phocytosis and monocytosis. There was a significant difference (F = 5733.96, df = 11,3, p < 0.001) between the plasma biochemical analysis of birds kept in the two wildlife hospitals. There were significant differences in the plasma concentrations of phosphorus (p < 0.001), potassium (p < 0.001), sodium (p = 0.18), albumin (p = 0.046) and globulin (p = 0.043) (Table 3). However, none of the plasma biochemistry results on ad- mission showed variation from reference ranges published for rowi and other kiwi (Morgan, 2008). It should be noted that the point- of-care analyser used has been criticised as being unreliable in its assessment of albumin and globulin (Greenacre et al., 2008) and the results should be interpreted with this in mind. 3.5. Radiography Full body radiographs in two orthogonal views of 12 birds taken in the first week following admission to hospital showed no de- tectable abnormalities. 3.6. Enteric bacteriology Cloacal swabs from 13 birds were negative on bacterial cul- tures for the presence of Salmonella spp and Yersinia spp. 3.7. Skin biopsies Histological examination of the biopsies of the affected skin and vent margins showed mild to moderate epidermal hyperplasia with hyperkeratosis in all eight samples examined. In 7/8 biopsies, there were also marked perivascular to nodular aggregates of lympho- cytes with fewer heterophils and eosinophils associated. In 5/8 biopsies, cross and tangential sections of nematode larvae were visible within the epidermis (Fig. 2). In several sections, there were intra-epidermal pustules comprised of small numbers of heterophils, Fig. 1. The gross appearance at the ventral abdomen and vent of two rowi on initial capture on the crèche island. In bird A, there is exudative dermatitis matting the feath- ers to the vent and uropygial gland. In bird B, there is alopecia and broken feathers of the skin around the vent and hyperkeratosis of the surrounding skin. Table 1 Faecal egg counts (eggs per gram of faeces) from rowi (Apteryx rowi) 1–3 days after admission to wildlife hos- pitals at Wildbase, Massey University and The Nest Te Kōhanga, Wellington Zoo. There are significant differ- ences between the two hospitals for the three types of parasite ova identified. The results are presented as means (±one standard error). Wildbase The Nest Te Kōhanga Coccidia 56,850 (±22,122) 1250 (±366) Capillaria 657 (±620) 19 (±13) Strongyles 6 (±3) 56 (±33) Table 2 Haematology from rowi (Apteryx rowi) 1–3 days after admission to wildlife hospitals at Wildbase, Massey University and The Nest, Wellington Zoo. Significant differences between birds kept in the different hospitals are highlighted in grey. (s.e. = one standard error). No eosinophils or basophils were detected in peripheral blood smears. No reference range is available for rowi. The reference range for brown kiwi (Apteryx mantelli) is from Morgan (2008). Wildbase The Nest Brown kiwi GLM stats Mean (±s.e.) Mean (±s.e.) Ref. range F df p Packed cell volume % 40.14 (1.62) 39.38 1.88 38–54 0.027 1 0.872 Total plasma solids (g/L) 40.00 2.89 43.83 2.88 0.872 1 0.370 WBCC (× 109 cells/L) 20.60 1.51 67.40 7.00 8.7–14.5 42.980 1 0.000 WBCC corrected (× 109 cells/L) 17.24 1.50 49.83 7.39 21.458 1 0.001 Heterophils (× 109 cells/L) 9.04 0.91 25.03 4.44 4.0–8.2 25.825 1 0.000 Lymphocytes (× 109 cells/L) 7.98 1.01 22.14 6.00 2.5–5.9 3.471 1 0.089 Monocytes (× 109 cells/L) 0.22 0.07 2.46 1.00 0.1–0.5 8.229 1 0.015 4 B.D. Gartrell et al./International Journal for Parasitology: Parasites and Wildlife 4 (2015) 1–10 eosinophils, multinucleated giant cells and small amounts of gran- ular necrotic debris. Microbial cultures from six of the biopsies were carried out through a commercial veterinary diagnostic laboratory. In the three birds from Wildbase that had cultures from the skin biopsies, aerobic bacterial cultures identified coagulase negative Staphylococcus aureus, Enterococcus sp., and Corynebacterium sp. in all three birds. Anaer- obic cultures of the skin biopsies grew low numbers of Clostridium perfringens. Fungal cultures were negative. Microbial cultures from the skin biopsies from the three birds from The Nest Te Kōhanga grew a greater diversity of micro- organisms. Aerobic bacterial cultures identified coagulase negative S. aureus, Enterococcus sp., Corynebacterium sp., Escherichia coli, Kleb- siella sp., and Proteus mirabilis. Fungal cultures were positive for Aspergillus fumigatus in all three biopsies and Fusarium solani complex in one biopsy. Three skin biopsies were dissolved overnight in pepsin/HCl and the digested material examined with a dissecting microscope for the presence of larvae; however, the skin failed to digest com- pletely, possibly because it had been fixed in 70% alcohol and no nematode larvae were able to be identified. Three mildly affected rowi that had not been previously sampled had skin biopsies four days after anthelmintic treatment as they showed increased pruritis and erythema of the ventral abdominal skin. Histological examination of these biopsies showed only mild epidermal hyperplasia and hyperkeratosis, with mild lymphocytic and eosinophilic dermatitis. 3.8. Molecular diagnostics Skin scrapings of the lesions from 10 birds were negative by PCR testing for the presence of herpesvirus, poxvirus and Chlamydia. 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http://refhub.elsevier.com/S2213-2244(14)00031-5/sr0170 http://refhub.elsevier.com/S2213-2244(14)00031-5/sr0170 http://refhub.elsevier.com/S2213-2244(14)00031-5/sr0175 http://refhub.elsevier.com/S2213-2244(14)00031-5/sr0175 http://refhub.elsevier.com/S2213-2244(14)00031-5/sr0175 http://refhub.elsevier.com/S2213-2244(14)00031-5/sr9010 http://refhub.elsevier.com/S2213-2244(14)00031-5/sr9010 http://refhub.elsevier.com/S2213-2244(14)00031-5/sr9010 http://refhub.elsevier.com/S2213-2244(14)00031-5/sr0180 http://refhub.elsevier.com/S2213-2244(14)00031-5/sr0180 http://refhub.elsevier.com/S2213-2244(14)00031-5/sr0180 http://refhub.elsevier.com/S2213-2244(14)00031-5/sr0185 http://refhub.elsevier.com/S2213-2244(14)00031-5/sr0185 Ventral dermatitis in rowi (Apteryx rowi) due to cutaneous larval migrans Introduction Materials and methods Physical examination Faecal parasitology Skin cytology Haematology and plasma biochemistry Radiography Enteric bacteriology Skin biopsies Molecular diagnostics PCR testing of skin scrapings for herpesvirus, poxvirus and Chlamydia PCR testing of skin biopsies for nematode sequences Sequencing of rDNA Phylogenetic analysis Post mortem examination Statistics Results Physical examination Faecal parasitology Skin cytology Haematology and plasma biochemistry Radiography Enteric bacteriology Skin biopsies Molecular diagnostics Complications of hospitalisation Discussion Cutaneous larval migrans Environmental factors contributing to infection Treatment and management of cutaneous larval migrans Aspergillosis Conclusions Acknowledgements Conflict of interest References