Longitudinal study of clinicopathological features of Johne's disease in sheep naturally exposed to Mycobacterium avium subspecies paratuberculosis.

The objective of this study was to describe chronological changes in infection status and enteric lesions of sheep naturally exposed to Mycobacterium avium subspecies paratubercuolosis. Samples of terminal ileum (TI) and mesenteric lymph node (MLN) were collected from 77 Merino sheep via surgical biopsy at 12, 18, and 24 months of age and necropsy at 36 months of age. Infection status at each sampling period was determined by fecal, TI, and MLN culture. Quantitative grading schemes were used to gauge the severity of granulomatous inflammation and degree of mycobacterial colonization affecting TI and MLN sections. Incidences of infection and disease were steady throughout the study; 46 of the 77 (59.7%) sheep became infected, and 30 of the 77 (39.0%) developed Johne's disease. Infection was first detected after 18 months of age in many sheep, and age when infection was first detected was not associated with clinical outcome. Culture of MLN detected 44 of the 46 (95.6%) infected sheep and initial lesions always involved MLN. Sheep typically developed lesions within 6-12 months following detection of infection by culture. The severity of enteritis and mycobacterial colonization progressed at variable rates among sheep. Severe multibacillary enteritis never regressed, and affected sheep expressed clinical signs within the following 12 months. Lymphocyte-rich paucibacillary enteritis was observed in 3 sheep, causing clinical signs in one and progressing to severe multibacillary enteritis in another. Six of the 46 (8.7%) biopsy-culture-positive sheep later had negative cultures at necropsy, suggesting recovery from infection. Further study is needed to identify factors associated with clearance of infection or progression of disease.

Association of microsatellite polymorphisms with immune responses to a killed Mycobacterium avium subsp. paratuberculosis vaccine in Merino sheep.

AIM: To study the association of polymorphisms at five microsatellite loci with immune responses to a killed Mycobacterium avium subsp. paratuberculosis (Map) vaccine. METHODS: Merino sheep (504 vaccinates and 430 unvaccinated controls) from a long-term Johne's vaccine trial undertaken on three different properties in the Central Tablelands of New South Wales, Australia, were genotyped for five microsatellite markers located in three immunologically significant chromosome regions. The marker loci included three from the major histocompatibility complex (MHC), namely DYMS1, OLADRB and SMHCC1; and one each from the solute carrier family 11 member 1 (SLC11A1), OVINRA1, and the interferon-γ (IFN-γ), o(IFN)-γ, gene regions. Associations between immune responses and genetic polymorphisms at the marker loci were examined by analysing both allelic and genotypic effects. RESULTS: The o(IFN)-γ locus had only two alleles, whereas the other four loci exhibited extensive polymorphism, with the number of alleles ranging from 10 (OVINRA1) to 21 (DYMS1), resulting in 30-92 genotypes per locus. Heterozygosities varied between 37% (o(IFN)-γ) and 87% (SMHCC1), while information on polymorphic contents ranged from 0.31 (o(IFN)-γ) to 0.87 (DYMS1). Each of the three properties exhibited unique allelic and genotypic frequencies. Analysis of immune response data revealed strong antibody and IFN-γ responses as early as 2 months post-vaccination. Immune responses in control animals on all three properties remained consistently low, except for slightly elevated IFN-γ responses at a few time-points on two properties, concomitant with exposure to natural infection. Genotype-phenotype association analyses revealed a number of marker genotypes/alleles to be significantly associated with antibody and IFN-γ responses. However, the effects of only five genotypes (one each at DYMS1, OLADRB, SMHCC1, OVINRA1 and o(IFN)-γ) and three alleles (one each at o(IFN)-γ, DYMS1and OLADRB) on IFN-γ responses were consistent across the three properties. CONCLUSION: Considering the significance of IFN-γ responses in protection against Map, it is possible that the genotypes/alleles identified might have a role in protective immune responses to natural Map infections, and further studies are warranted to confirm this.

A survey to detect the presence of Mycobacterium avium subspecies paratuberculosis in Kangaroo Island macropods.

The aim of this study was to determine whether Mycobacterium avium subspecies paratuberculosis (M. a. paratuberculosis) infection was present in macropods grazing with infected sheep on Kangaroo Island in 2001-2002, and to assess the likely role of such infection in the epidemiology of ovine paratuberculosis. Ileum and associated lymphatics from 482 macropods were examined using radiometric culture followed by PCR for IS900 and restriction endonuclease analysis (REA) for species identification, and isolates were strain typed using PCR for IS1311 and REA. Ileum and mesenteric lymph nodes from animals with positive tissue cultures or gross lesions suggestive of paratuberculosis were examined histologically. Faeces from a total of 840 animals were cultured in pools of 20, and individual faecal cultures were done from tissue culture positive animals, from those with microscopic lesions, and from selected animals with gross lesions. Eight animals (1.7%) yielded positive tissue cultures, and all isolates were the sheep (S) strain. Two animals that were tissue culture positive also had histopathological evidence of paratuberculosis. Twelve culture negative animals had microscopic lesions consistent with mycobacterial infection, and M. genavense was identified by PCR from a paraffin block from one of these animals. All faecal cultures were negative. These results indicate that a small proportion of macropods can become infected with M. a. paratuberculosis when grazing with infected sheep. However, excretion of large numbers of viable organisms is rare in macropods, and it is unlikely that macropods provide a wildlife reservoir of infection that would seriously compromise control efforts for paratuberculosis in sheep.

Isolation of Mycobacterium avium subspecies paratuberculosis from muscle and peripheral lymph nodes using acid-pepsin digest prior to BACTEC culture.

Meat has received little attention regarding human exposure to Mycobacterium avium subsp. paratuberculosis, a possible infectious trigger of Crohn's disease. Meat has less contamination with other organisms than gut tissues, facilitating modifications to existing decontamination protocols prior to BACTEC culture that could increase analytical sensitivity. Using spiked meat samples we trialled enzymatic and chemical digestion techniques to concentrate larger starting samples, and modifications to existing clinical mycobacteriological decontamination protocols. An acid-pepsin digestion method using a 20 g sample was considerably more sensitive (detection limit 0.88 log(10) viable organisms per gram) than previous techniques. However, it was cumbersome for routine use, and subject to frequent contamination. Modifications to an existing centrifugation protocol yielded a simple, robust technique with slightly improved sensitivity (detection limit 1.77 log(10) per gram). Use of these sensitive tests in parallel identified M. a. paratuberculosis in the muscle of 59% and peripheral lymph nodes (PLN) of 85% of clinically infected sheep. The numbers of M. a. paratuberculosis in these infected tissues were low (1.67+/-0.92 log(10) per gram in muscle and 2.06+/-0.69 log(10) per gram in PLN), such that many would not have been detected by routine methods. Fewer subclinically infected animals with gross lesions harboured M. a. paratuberculosis in meat (4.5%) or PLN (32%), and the numbers of organisms in such infected animals were lower. Because most animals raised specifically for meat production are young and unlikely to be heavily infected, and because meat is usually consumed cooked, the risk of human exposure to viable M. a. paratuberculosis via meat may be small. Measures to prevent heavily infected animals, especially those with clinical signs, from entering the human food chain would further reduce this risk.

Multiplex PCR for the detection of Brucella ovis, Actinobacillus seminis and Histophilus somni in ram semen.

OBJECTIVE: To develop a multiplex polymerase chain reaction (PCR) assay for the rapid detection of Brucella ovis, Actinobacillus seminis, Histophilus somni in fresh ram semen samples. DESIGN: The multiplex assay was based on the single PCR assays published for the detection of A seminis and B ovis, and the forward primer published for the detection of H somni; an alternative reverse primer for H somni was designed in this study. PROCEDURE: Culture and PCR of 295 fresh semen samples were carried out. RESULTS: The multiplex PCR was far more successful in the detection of H somni (45/295) than culture (23/295). A seminis was also detected in more semen samples by multiplex PCR (29/295) than culture (13/295) and B ovis was detected in three samples using both PCR and culture. No amplifications were detected with DNA from a range of bacterial isolates including species associated with epididymitis in rams. CONCLUSION: This PCR could be used as a complementary test, or alternative to culture of ram semen and other biological samples for the detection B ovis, H somni and A seminis.

Efficacy of a killed vaccine for the control of paratuberculosis in Australian sheep flocks.

A field trial was undertaken from 1999 until 2004 to determine the efficacy of a killed M. a. paratuberculosis vaccine, Gudair, for the control of ovine Johne's disease (OJD) in merino sheep run under Australian pastoral conditions. On each of three farms experiencing significant OJD losses (5-15% per annum), 200 merino lambs (age 1-4 months) were vaccinated, and 200 lambs were left unvaccinated. Animal assessments and sample collections were conducted twice yearly until 4 or 5 years of age. The impact of vaccination on mortality rate, faecal shedding of M. a. paratuberculosis (by pooled and individual faecal culture), liveweight, wool productivity, vaccine injection site lesions and cellular (BOVIGAM) and humoral (PARACHEK) immunity was examined. Vaccination stimulated cell-mediated and humoral immune responses, reduced mortalities due to OJD by 90% and delayed faecal shedding for the first year post-vaccination. Thereafter, the prevalence of shedders among vaccinates was reduced by 90%. The numbers of M. a. paratuberculosis excreted by the vaccinated groups were also reduced by at least 90% at most sampling times. However, high levels of excretion by vaccinates occurred on some occasions, and although only 7 of 600 vaccinates died from OJD, all 7 had multibacillary lesions. Thus there remains a risk that some vaccinated sheep will transfer the disease. Small reductions in liveweight were found in vaccinated lambs in the first year, but there was little effect on wool production. Vaccine injection site lesions were detected in almost 50% of sheep after 2 months, and these persisted for at least 4 years in 20-25% of vaccinates. Data from this trial enabled the registration of Gudair in Australia in 2002 and underpins the pivotal role of vaccination in the current management of OJD.

A preliminary study of possible genetic influences on the susceptibility of sheep to Johne's disease.

OBJECTIVE: To investigate possible genetic influences on susceptibility or resistance of sheep to Johne's disease. DESIGN: A field and laboratory study of two fine-wool Merino flocks with a high prevalence of disease due to Mycobacterium avium subsp paratuberculosis infection. PROCEDURE: Adult sheep were phenotypically classified as having severe, mild or no disease on the basis of clinical, pathological and cultural tests for paratuberculosis, and as positive or negative in tests for humoral immunity (agar gel immunodiffusion test) or cell mediated immunity (skin test for delayed type hypersensitivity). Correlations with phenotype were sought for polymorphisms at loci within selected immune function genes (NRAMP, MHC complex, IFN-gamma, lysozyme, leukaemia inhibiting factor). RESULTS: Possible associations of particular NRAMP and MHC alleles with susceptibility or resistance to Johne's disease were detected. CONCLUSION: If the results of this preliminary study are confirmed in further work, then the use of rams with "resistant" genotypes may assist in the control of Johne's disease in infected flocks.

Intrauterine and transmammary transmission of Mycobacterium avium subsp paratuberculosis in sheep.

OBJECTIVE: To investigate intrauterine infection of foetuses with Mycobacterium avium subsp paratuberculosis and the presence of infection in mammary secretions of sheep. DESIGN: A study of 142 late-pregnant ewes and their foetuses from two heavily infected flocks. PROCEDURE: Infection of ewes was determined at necropsy by histopathology and culture of tissues and mammary secretions. Antemortem tests (clinical assessment, faecal culture and serology) were also applied. Foetuses from 59 infected ewes and 47 apparently uninfected ewes were examined by culture and histopathology. RESULTS: Five of five ewes with clinical ovine Johne's disease had infected foetuses. Only one of 54 subclinically affected ewes, and none of 47 uninfected ewes had an infected foetus. M a paratuberculosis was cultured from mammary secretions or mammary glands of only two of 76 ewes, both of which were clinical cases and had infected foetuses. CONCLUSION: Although intrauterine or transmammary transmission of Mycobacterium avium subsp paratuberculosis may occur frequently in clinically affected sheep, these are less common in subclinically infected ewes. Therefore these modes of transmission are unlikely to compromise existing control programs for ovine Johne's disease on most farms, especially if programs include the immediate culling of clinically affected sheep.

Field evaluation of tracer sheep for the detection of early natural infection with Mycobacterium avium subsp paratuberculosis.

OBJECTIVE: To determine whether tracer sheep could be used to detect S strain Mycobacterium avium subsp paratuberculosis on pasture, and to provide further insight into the early stages of infection. DESIGN: A field study on two farms in an endemic area for ovine Johne's disease in New South Wales. Procedure Lambs, weaners and adult ewes were introduced to pasture with varying amounts of M. a. paratuberculosis contamination and monitored using skin tests, gamma interferon assay, faecal culture and serial necropsy of small groups for up to 15 months after first exposure. RESULTS: Culture from tissues was the most sensitive method for detecting early infection in sheep after natural exposure to S strain M. a. paratuberculosis. The organism was detected in at least one introduced sheep from every exposed group, 6 to 12 months after first exposure. Histopathological lesions were detected in only 17% of culture-positive sheep, and only after at least 8 months of exposure. Similarly, antemortem diagnostic tests had low sensitivity during the early stages of naturally acquired infection. There was no evidence of any differences in infection rate between sheep first exposed as neonates, as weaners or as adults. A higher proportion of lambs born to ewes from an infected flock were infected than lambs suckling uninfected ewes introduced to the same infected environment, and infection was detected earlier in these 'resident' lambs. CONCLUSION: These findings indicate that groups of unexposed 'tracer' sheep, tested by culture of tissues at slaughter 6 to 12 months after first exposure, might be a useful way to assess pasture infectivity in control programs for ovine Johne's disease.

Immunoperoxidase studies of cell mediated immune effector cell populations in early Mycobacterium avium subsp. paratuberculosis infection in sheep.

Immunoperoxidase (IPX) labelling for CD4, CD8, TCR-gammadelta, WC1, CD1b, IFN-gamma, CD45R, CD56 and lysozyme was used to investigate changes in cell mediated immune effector cell populations in the intestinal Peyer's patches (PP) and mesenteric lymph nodes of lambs, 2 and 4 months after experimental infection with low doses of sheep strain Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis). The organism was cultured from the tissues of each infected lamb, but histological lesions were not present. This infection model was considered to be more representative of natural M. a. paratuberculosis infection than previous studies. Infected sheep had significantly more CD4+ cells in the mucosa, domes and interfollicular areas of the terminal ileum, and in the interfollicular areas of the jejunal Peyer's patch. Infected sheep also had significantly increased numbers of TCR-gammadelta+ cells in the mucosa and interfollicular areas of the jejunal Peyer's patch, and increased numbers of WC1+ cells in the ileal Peyer's patch. These findings are consistent with previous findings in sheep given higher doses of cattle strain M. a. paratuberculosis. Significantly fewer CD1b+ cells were present in the paracortical areas of the mesenteric lymph nodes of infected sheep, and the reduction was greater in sheep infected for 4 months compared to sheep infected for only 2 months. Down-regulation of CD1b expression may be important for the continued survival and multiplication of M. a. paratuberculosis as specific adaptive immunity develops. Across all sheep, jejunal Peyer's patches had higher numbers of CD4+, CD8+, TCR-gammadelta+, WC1+ and CD45R+ cells, and lower numbers of CD56+ fibres compared to ileal Peyer's patches. These findings confirm and extend the peculiarities of the terminal ileal Peyer's patch in the young ruminant, with possible implications for the early establishment of M. a. paratuberculosis infection.

Experimental infection of weaner sheep with S strain Mycobacterium avium subsp. paratuberculosis.

We sought to determine whether infection of recently weaned 12-16-week-old Merino lambs with an Australian S strain M. a. paratuberculosis, at doses consistent with natural exposure, could be detected in the first few months post-inoculation. Such detection would facilitate the use of weaner sheep as sentinel animals for the presence of infectious doses of M. a. paratuberculosis on pastures. In controlled pen trials, oral doses of approximately 10(7)-10(8) viable organisms were demonstrated to be infective, whereas doses below 10(4) organisms failed to produce detectable infection. Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) was isolated from intestinal and/or lymphoid tissues collected at necropsy 7 or 14 weeks after first infection, but there were no associated gross or microscopic lesions. Skin testing with intradermal Johnin detected all three infected lambs at 13 weeks post-infection, and one of the three infected lambs at 6 weeks post-infection, with 100% specificity. Results for whole blood IFN-gamma assay showed some correlation with infection status but lacked specificity. One infected lamb gave a positive result in an ELISA for antibodies to M. a. paratuberculosis, 14 weeks post-infection and 1 week after skin testing. This was the first demonstration of experimental infection with S strain M. a. paratuberculosis in Australian Merino sheep at doses likely to be representative of natural infection. Culture from tissues in the first few months post-exposure could facilitate the use of naive weaner sheep as tracer animals to detect heavy contamination of pastures with M. a. paratuberculosis, but low-level contamination may not be detected in such a system.

The effect of decontamination protocols on the numbers of sheep strain Mycobacterium avium subsp. paratuberculosis isolated from tissues and faeces.

The effect of decontamination protocols on the numbers of sheep strain Mycobacterium avium subsp. paratuberculosis isolated in BACTEC cultures from clinical samples was assessed by spiking tissues and faeces at various points during the decontamination procedure. Routine protocols in the laboratory were shown to decrease the number of organisms isolated per sample by about 2.7 log(10) and 3.1 log(10) for faeces and tissues, respectively. These findings are important for the interpretation of negative culture results and may be useful in epidemiological studies. Addition of a centrifugation step to the tissue protocol increased the recovery by about 1 log, but resulted in increased contamination of BACTEC cultures. These studies may also facilitate future improvements to decontamination procedures.

Isolation of Mycobacterium avium subsp paratuberculosis from environmental samples collected from farms before and after destocking sheep with paratuberculosis.

OBJECTIVE: To determine whether Mycobacterium avium subsp paratuberculosis could be isolated from soil-pasture, faecal, water and sediment samples on farms before and after removal of sheep with paratuberculosis. A feasibility study and subsequent field survey. PROCEDURE: First the analytical sensitivity of radiometric culture of the organism from two types of soil was determined relative to faeces. Then soil-pasture, faecal, water and sediment samples were collected for culture from a range of sites from 6 farms with paratuberculosis affected sheep and goats. Similar samples were collected from 20 farms at least 9 months after removal of infected stock. RESULTS: The analytical sensitivity of culture of M a paratuberculosis from soil samples was 2 orders of magnitude less than that from faeces, and environmental samples required longer incubation periods to yield significant growth in radiometric culture (BACTEC) medium. However, the organism was recovered from approximately 20% of 163 soil-pasture, water and sediment samples from 6 properties with clinically-affected animals with paratuberculosis. The positive samples were from a range of topographic sites, including open exposed and dry areas, however, low lying areas tended to have larger numbers of organisms. When the same sites were sampled again about 5 months later, only 1 was culture positive, and none were culture positive > 12 months later. Of 17 water and dam sediment samples collected from farm 6, which had long-standing high prevalence OJD infection, only one water sample and one sediment from the same dam were culture positive. None of the 5 water samples from the other farms were culture positive. Of 96 water samples, 90 sediment samples and 93 soil samples from farms that had been destocked of infected sheep/goats for 9 to 24 months, one sediment sample from a farm in Victoria (destocked for 12 months) and two sediment samples from a farm in New South Wales (10, 19 months) were culture positive. Recontamination from cattle or water could not be excluded as a cause of the positive cultures from the second farm. CONCLUSION: M a paratuberculosis can be detected by radiometric culture in environmental samples from farms grazed by sheep or goats with paratuberculosis. There is a relatively low likelihood of recovery of the organism from water samples from such farms, and at 5 or more months after removing stock with paratuberculosis the likelihood of positive cultures from environmental samples is very low. Although the analytical sensitivity of culture from environmental samples is less than that from faeces, surveys of environmental sites are nevertheless feasible. However, improved culture methods are needed for critical surveys and to study the movement and fate of the organism in the environment.

Recovery of Mycobacterium avium subsp. paratuberculosis from nematode larvae cultured from the faeces of sheep with Johne's disease.

A study was conducted to determine whether trichostrongylid nematode larvae become contaminated with Mycobacterium avium subsp. paratuberculosis when they develop in the faeces of sheep with Johne's disease. Nematode larvae were hatched from ova in the faecal samples of affected sheep. Larval sheaths were removed and these as well as exsheathed larvae were subjected to radiometric culture for M. paratuberculosis. The organism was recovered from washing water used to prepare the larvae, third stage larvae and larval sheaths, but not from exsheathed larvae. The recovery of M. paratuberculosis from larvae was associated with the severity of the histological lesions in affected sheep and with the results of culture of the organism from intestinal tissues and faeces. Nematode parasites of sheep might be able to act as mechanical vectors for M. paratuberculosis as the organism associates with infective third stage larvae when these develop in the faeces of sheep with Johne's disease.

Seroprevalence of Neospora caninum infection following an abortion outbreak in a dairy cattle herd.

OBJECTIVE: To investigate the seroprevalence of Neospora caninum infection in a commercial dairy cattle herd, 15 months after detection of an abortion outbreak. PROCEDURE: Sera from the whole herd (n = 266) were examined for N caninum antibodies by indirect fluorescent antibody test (IFAT) and immunoblot analysis. Herd records were reviewed to collate serological results with abortion history, proximity to calving, and pedigree data. RESULTS: The seroprevalence of N caninum infection was 24% (63/266) for IFAT titre > or = 160, 29% (78/266) for immunoblot positive (+ve), and 31% (82/266) for IFAT > or = 160 and/or immunoblot +ve; 94% (59/63) of animals with IFAT > or = 160 were immunoblot +ve. The association between seropositivity (IFAT > or = 160 and/or immunoblot +ve) and history of abortion was highly significant (P < 0.001); the seroprevalence was 86% (18/21) in aborting cows, compared with 30% (50/164) in non-aborting animals. The abortion rate for seropositive cows was 26% (18/68) compared with 3% (3/117) for seronegative animals. IFAT titres of infected cows were higher within 2 months of calving than at other times (P < 0.001). The association between seropositivity in dams and daughters was highly significant (P = 0.009). CONCLUSIONS: The abortions were associated with N caninum infection and there was evidence of reactivation of latent infection close to calving and congenital transmission of infection. Immunodominant antigens identified by immunoblots may prove useful for improved diagnostic tests.

Temporal patterns and quantification of excretion of Mycobacterium avium subsp paratuberculosis in sheep with Johne's disease.

OBJECTIVES: To determine the frequency of excretion of Mycobacterium avium subsp paratuberculosis in Merino sheep with Johne's disease and to quantify excretion in a group of Merino sheep. DESIGN: A pen and laboratory experiment. PROCEDURE: Seven sheep selected from an affected flock on the basis of acid-fast bacilli in the sheep's faeces were housed and total daily faecal output was collected, weighed and subjected to culture for M avium subsp paratuberculosis. An end-point titration method was used to enumerate viable M avium subsp paratuberculosis in a 15 day pooled sample from five sheep that had acid-fast bacilli in their faeces while housed. RESULTS: Four sheep with subclinical multibacillary Johne's disease excreted M avium subsp paratuberculosis each day for 11 days of cultural observation. A further three sheep were intermittent excreters but lacked other evidence of infection with M avium subsp paratuberculosis. The average number of viable bacteria excreted was 1.09 x 10(8) per gram of faeces while total daily excretion was 8.36 x 10(10) viable M avium subsp paratuberculosis per sheep. Examination of faecal smears stained with Ziehl Neelsen was an unreliable means of assessing daily excretion in individual animals except in those with severe lesions. CONCLUSION: Excretion of M avium subsp paratuberculosis in Merino sheep with multibacillary Johne's disease occurred daily, proving that environmental contamination can be continuous on farms with endemic ovine Johne's disease. Faecal culture is a useful method for detecting infection as it does not appear to be affected by the timing of collection of a sample from sheep with multibacillary disease however, to maximise the sensitivity of disease surveillance using faecal culture, sampling rates should be adjusted to take account of the proportions of multibacillary and paucibacillary cases.

Experimental reproduction of viral chorioretinitis in kangaroos.

OBJECTIVE: To investigate whether preparations containing Wallal and/or Warrego viruses could cause disease when inoculated subcutaneously into captive kangaroos. DESIGN AND PROCEDURE: Four groups of two kangaroos, seronegative to both Wallal and Warrego virus, were each inoculated with wild Wallal virus, cultured Wallal virus, wild Warrego virus, or wild Warrego virus followed by wild Wallal virus after 3 weeks. A single uninoculated animal served as a control. Animals were monitored weekly under anaesthesia, examined ophthalmoscopically (including fundic photography), and samples collected for haematological and serum biochemical analysis, virus isolation, PCR and serological examination for antibodies against Wallal and Warrego viruses. Animals inoculated with cultured Wallal virus were killed at week 10, and remaining kangaroos were reinoculated with cultured Wallal virus at week 12. RESULTS: Virus was isolated from the blood of two kangaroos 2 weeks after inoculation with Wallal virus preparations, and from a third kangaroo 2 weeks after reinoculation. By 3 weeks after inoculation, all kangaroos given Wallal virus preparations had seroconverted to Wallal virus and one had seroconverted to Warrego virus. Fundic changes were detected in the three viraemic kangaroos 4 or more weeks after inoculation, and lesions were present in the eye and brain typical of those seen in field cases of chorioretinitis. No other kangaroos had lesions. Wallal virus was identified by PCR and immunohistochemical analysis in the retina of one affected animal and orbivirus-like particles were seen by electron microscopy in the remains of retinal cells. CONCLUSION: The condition of chorioretinitis was reproduced in three of eight kangaroos by inoculation with preparations containing Wallal virus.