key: cord-0010590-52mlkdc6 authors: WYLIE, SL.; PASS, DA. title: Investigations of an enteric infection of cockatoos caused by an enterovirus‐like agent date: 2008-03-10 journal: Aust Vet J DOI: 10.1111/j.1751-0813.1989.tb09716.x sha: 7af32470d0e922349943d9a0b39fa4c9c14793f5 doc_id: 10590 cord_uid: 52mlkdc6 An enteric infection in cockatoos associated with a 30nm diameter enterovirus‐like agent seen in faeces and intestinal epithelial cells is described. The disease is characterised by intractable, profuse, mucoid diarrhoea, weight loss, dehydration and death. Lesions in the intestine consist of villous atrophy, villous fusion, enterocyte hyperplasia and, in some cases, chronic inflammation. Affected birds so far examined have concurrent psittacine beak and feather disease. at laboratories. Evaluation of infection status on the basis of a single isolate per swab rather than multiple isolates may also have contributed t o differences. Our study showed a significant crude association between purchase of more than 5 pigs from herd SA and isolation of toxigenic P. multocida. Of 3 herds in this category, the 2 that purchased the greatest number of breeding pigs (9 and 13) from herd SA, were classed as infected. The probability of introducing infected pigs is a function of prevalence of infection in the source herd and the number of introductions. For example, if 10% of pigs in herd SA were infected, the probabilities of these 2 herds not introducing infection ((1-P)") were 0.39 and 0.25, respectively. Owners of both infected herds in the high risk group also introduced more than 50 pigs from other sources in the 2 years prior to the study but this factor was unlikely to be causal. All other herds except one, which supplied pigs to the 3 positive herds in the 2 years prior to the survey were sampled in a similar fashion and found free from clinical or cultural evidence of infection. Samples were not collected from one source, a large breeding company, but the consulting veterinarian indicated that the herd was free from infection (B Munro, personal communication 1988) . Therefore, we believe that introduction of more than 5 pigs from herd SA was causally associated with the risk of isolation of toxigenic P. multocida and that the observed statistical association between introduction of more than 50 pigs from other sources and isolation was a spurious one. Evaluation of factors other than introductions was limited by the small number of herds from which toxigenic isolates were recovered and the small number of herds in some exposure categories. Smith (1983) reported that management and housing factors such as poor ventilation, continuous throughput in farrowing and weaner accommodation, and high stocking densities were important risk factors for atrophic rhinitis. Such evaluations will be more appropriate should atrophic rhinitis become more prevalent in NSW. Enteric disease associated with the presence of virus particles in faeces is becoming more widely recognised in birds. Reynolds and Saif 1986) ; parvoviruses in chickens (Kisary 1985) and turkeys (Trampal et This paper describes the clinical signs and lesions of an enteric disease in galahs (Cacatua roseicapi//a) and a sulphurcrested cockatoo (C. galerita) associated with the presence of virus particles in the faeces. cases. The clinical and pathological description is based on 17 The disease was first seen in 6 young galahs in a flock of wild birds in the outer metropolitan area of Perth, Western Australia. The flock is composed largely of aviary escapees as many birds have the physical characteristics of galahs originating in the eastern states of Australia, ie. they have much paler feathers on the crown than birds from the southwest of Western Australia. (D Saunders CSIRO, Division of Wildlife and Ecology). Numerous birds in the flock also had psittacine beak and feather disease (PBFD) (Pass and Perry 1984) and all birds subsequently examined by us that have had enteric viral infection have been diagnosed as having PBFD concurrently. Six cases were caged birds held by us for experimental purposes and the remainder were "pets" recently purchased from bird dealers. Faeces from some affected birds were cultured on sheep blood agar and MacConkey's agar and were also examined by nega .ve contrast electron microscopy. Faeces were centrifuged to remove solid debris and a drop of the supernatant was placed on a formvar coated copper grid, stained with 3% phosphotungstic acid (pH 7.2) for 1 min and then examined in a Philips 301 electron microscope. Necropsies were performed on birds that died or were killed humanely with intravenous barbiturate. Representative areas of intestine were destroyed, in cold 3% glutaraldehyde in 0. I M phosphate buffer (pH 7.2). Skin, liver, kidney and bursa of Fabricius were fixed in 10% buffered formalin. Sections for both light and electron microscopy were processed routinely. Frozen sections of formalin-fixed intestine were stained for lipid with oil red 0. Blood was collected from four affected birds and the serum examined for the presence of antibodies to human, chicken, feline, canine and bovine caliciviruses and to human astroviruses 1, 2 and 4 by Dr D Cubitt, Public Health Laboratory Service, Central Middlesex Hospital, London. Figure 1 . Virus-like particles in faeces. Bar = O.lpm. AttemDts were made to transmit the infection bv oral inoculatio; to 6 normal galahs, 2 galahs with PBFD,*4 peachfaced lovebirds (Agapornis roseicollis) with PBFD and 4 oneday-old SPF chickens. The inoculum used was faeces that contained virus. The faeces which had been stored at -2OOC were clarified by centrifugation and the supernatant administered orally via a crop tube on 3 successive days. SPF chickens, Agapornis sp and galahs received 0.5, 2.0 and 3.0ml of inoculum respectively. The birds were kept for 21 days after inoculation. Faeces were examined for the presence of viruses by negative contrast election microscopy before infection, daily for 7d, and every 2 to 3d thereafter. The major presenting clinical sign is profuse, green, mucoid diarrhoea. Depression and poor appetite of variable severity are apparent in most cases presented by owners. Our observations on the initial cases diagnosed in wild galahs suggests that depression and poor appetite are not apparent, or very mild, in the first few days after the onset of diarrhoea, but as the diarrhoea continues, these signs increase in severity. Weight loss and dehydration rapidly ensue. The diarrhoea is persistent and all but one of the affected galahs that we have seen have died. The course of the disease varies from several days to 3 or more weeks and at the time of death the bird is emaciated. In one case, diarrhoea was present for 4d and the bird recovered spontaneously. The history of this bird differed from the others. It was diagnosed as having zinc poisoning from ingestation of galvanised wire and was treated successfully but developed diarrhoea 4d after treatment for zinc toxicity was completed. This bird also had PBFD. The disease spreads rapidly to other birds. Two affected birds were placed in a room containing individually caged galahs and sulphur-crested cockatoos suffering from PBFD. Figure 2 . Viscera of an affected bird. Note the dilated The enteric disease occurred within 5d in 5 galahs and one 322 intestine. sulphur-crested cockatoo that previously had no evidence of diarrhoea. Diarrhoea was less severe in the sulphur-crested cockatoo than in the galahs. Five of the resident galahs died but the sulphur-crested cockatoo survived. Negative contrast electron microscopy of faeces from affected birds revealed large numbers of round unenveloped virus particles approximately 35nm in diameter (Figure 1 ). The particles do not have any definite surface structure. Grossly, the intestine is dilated with mucoid fluid and gas and the wall is thickened (Figure 2) . Microscopically, lesions are visible along the entire length of the small intestine but are most noticeable in the duodenum and upper jejunum. Lesions consist of villous atrophy, fusion of villi, elongation of crypts of Leiberkuhn and hyperplasia of epithelial cells in crypts and on shortened villi (Figure 3) . Enterocytes on villi are columnar and those on the tips of villi are often vacuolated. Vacuoles contain lipid. lnflammation of variable severity may be present. In some areas, this may only be an increase in plasma cells and lymphocytes in the lamina propria but in other areas heterophils and macrophages that contain eosinophilic granular material or haemosiderin are prominent. Acute inflammation and haemorrhage into the villous tips has been seen in some cases. Overall, the mucosa is thickened despite villous atrophy. Large numbers of bacteria are commonly present in the lumen of the gut and a heavy growth of bacteria may be cultured from faeces. Transmission electron microscopy of the intestinal mucosa of one affected bird revealed paracrystalline arrays of round virus-like particles approximately 30nm in diameter in the cytoplasm of enterocytes (Figure 4) . Many enterocytes had less numerous and shorter microvilli than normal. Attempts to experimentally infect SPF chickens, Agapornis sp, normal galahs and galahs with PBFD failed. Virus was not detected in faeces of any bird. Sera from naturally affected galahs and experimentally infected SPF chickens and Agapornis sp did not contain detectable antibodies against human, canine, feline, bovine or chicken Caliciviruses or Human Astroviruses 1, 2 and 4. The presence of virus-like particles in faeces and in the cytoplasm of enterocytes of the small intestine strongly suggest that the birds described in this report were suffering from an enteric viral infection. This is supported by the type of lesion present in the small intestine. Villous atrophy and fusion, and crypt cell hyperplasia indicate the enterocytes on the villi have been destroyed, a process typical of some viral infections, particularly coronaviruses and rotaviruses (Cheville, 1983) . Although it remains unproven, we believe that the virus-like particles seen in faeces are the most probable cause of the disease. The morphology of the virus-like particles in faeces and enterocytes closely resemble those described in an entero-like virus infection in chickens (McNulty et a1 1985) . The association of this disease with PBFD suggests that it occurs in birds with reduced immunological competence. our observations on the presence of PBFD virus in the bursa of Fabricius and thymus together with the fact that affected cockatoos often develop acute bacterial infections, cryptosporidial infection of the lower gut and myeloproliferative diseases suggests that some of these birds d o suffer some form of immunodepression. Our initial experience with the disease indicated that it is very infectious as it rapidly spread to captive birds with PBFD after the first cases were introduced to the bird room. We were unable, however, to experimentally infect birds under similar conditions at a later date. The reasons for this are not known. It is possible that the birds used were not susceptible to infection because of past exposure to the virus o r that the inoculum used was non-infectious even though virus particles were plentiful. Storage of faeces at -2OoC, however, may have reduced infectivity of the virus. Haematological and biochemical analyses provide a useful adjunct to the diagnosis of disease in domestic animals. It has been assumed that a similar situation applies to wildlife, especially for those animals kept in zoos or sanctuaries. However, before the usefulness of these analyses for the diagnosis of disease can be assessed, reference (normal) values have to be established. Ideally this should be done for each population with a different habitat, but in practice values are extrapolated from one group to another. In the koala, apart from unpublished data available from zoos, limited reference values are available. Two studies have been published for various wild groups from Victoria (Obendorf 1983) and New South Wales (NSW) (Dickens 1975) . In this article reference values are established for a population of free living koalas centred around Port Macquarie on the north coast of NSW. This population is under threat because of urbanisation and a high level of natural occurring disease (Canfield 1987). Consequently the Koala Preservation Society of NSW and local veterinarians are utilising haematological and biochemical analyses for diagnosis and prognosis. Forty-five apparently healthy koalas were used. These animals were captured at various times during the year for the purposes of relocation. They comprised 16 adult females, 13 adult males, 15 juvenile males and one juvenile female. For the purposes of this study adults were defined as greater than 2 to 3 years of age (based on tagging records and dentition). Juveniles were predominantly 1 to 2 years of age. Koalas were physically restrained in a hessian bag and bled either at the time of capture or prior to release. The period of capture was usually less than 12 h. Venous blood was 324 collected from the forearm and placed in a plain serum tube and a tube containing sequestrene (EDTA). Serum was separated and a peripheral blood film prepared within one hour of collection. The samples were then transported at 4°C to a laboratory, where they were processed within 12 h of collection. Biochemical testing was done using a Hitachi 737*, a discrete, selective sample oriented analyser. Boehringer Mannheim reagents* were used for all tests except for calcium? and biocarbonatet. Sodium, potassium and chloride analyses involved ion specific electrodes, albumin analysis utilised bromcresol green while total protein analysis involved the Biuret method. Globulins were derived from the difference between total protein and albumin. Enzyme analyses were all performed at 37°C. Anionic gap was determined by the following formula: (sodium + potassium) -(chloride + biocarbonate). Osmolality was estimated from 2 x (sodium + potassium). Haematological analysis (haematocrit, haemoglobin, erythrocytes, leukocytes and platelets) was performed on a Coulter S 880$. A peripheral blood film was stained with Diff Quiks and manually assessed for morphology and leukocycte types. Values were analysed using Minitab( and Genstat# statistical packages on an IBM personal computer. Parametric statistics were applied after assessment of normality by probability plots (N scores -Ryan et al 1985) . In some instances square root transformations were required while in 2 (monocytes and eosinophils) no transformations could produce a normal distribution due to their limited range. Significant differences between ages, sexes and seasons were determined using anal- Cell pathology Diseases of Poultry The authors gratefully acknowledge the co-operation of piggery owners and the assistance of Pastures Protection Board and Department of Agriculture and Fisheries veterinarians with farm visits. Staff at Regional Veterinary Laboratories kindly cultured nasal swabs and identified P. multocida isolates. H Ridings provided valuable assistance with the statistical nalysis. The study was financially supported by the Swine Compensation Fund of New South Wales.