Rapid diagnosis of highly pathogenic avian influenza using pancreatic impression smears.

The 1985 outbreak of high-pathogenicity avian influenza (HPAI) in Victoria, Australia, took 5 days to confirm by standard laboratory tests, during which time infected chickens continued excreting virus, thus creating the opportunity for transmission to other farms. An immunofluorescence test for the detection of viral antigen in tissue impression smears was evaluated as a rapid diagnostic test for HPAI virus infections of poultry. Several test configurations were compared for background reactions and strength of fluorescence, with the optimum combination found to be an influenza A group-specific monoclonal antibody, detected by an anti-mouse fluorescein isothiocyanate conjugate. Immunohistochemical examination of tissues from chickens experimentally infected with low-pathogenicity and HPAI viruses identified the pancreas as the organ most consistently containing high concentrations of HPAI viral antigen. This test has since been used in Australia in the rapid laboratory confirmation of three avian influenza outbreaks and in showing that numerous other suspect cases were not caused by avian influenza.

A guinea-pig model of Hendra virus encephalitis.

Subcutaneous inoculation, but not intradermal (footpad) or intranasal inoculation, with high doses of Hendra virus (HeV) consistently produced disease in guinea-pigs. Of 15 subcutaneously inoculated animals, 14 developed vascular disease with positive HeV immunohistochemical labelling in a range of tissues. A new observation was the presence of lesions, including syncytial cells, with immunolabelling in the transitional epithelium of the bladder. Virus isolation from the urine rather than from nasal, oral, rectal or conjunctival swabs, the other external sites, was consistent with previous epidemiological work in horses, indicating a limited possibility of transmission. The dose used (30 000 to 50 000 TCID(50)), which was higher than in previous studies, produced microscopical lesions of encephalitis in eight of the 15 subcutaneously inoculated guinea-pigs, with positive immunolabelling in blood vessels and neurons, especially in the medulla, cerebellum and thalamus. The virus was recovered from six of the encephalitic brains. Severe vascular degeneration in the centres of encephalitic lesions in six of the eight encephalitic guinea-pigs and positive immunolabelling in the choroid plexus of a further animal indicated that the virus entered the brain following virus-induced vascular injury and choroid plexus invasion. Guinea-pigs would appear to be suitable for the study of HeV encephalitis.

Vaccination of pigs with a recombinant porcine adenovirus expressing the gD gene from pseudorabies virus.

Five week old, commercially available large white pigs were vaccinated with either a single dose or two doses of a recombinant porcine adenovirus expressing the glycoprotein D gene from pseudorabies virus (PRV). Pigs were monitored for the development of serum neutralizing antibodies to PRV and challenged 3 weeks after final vaccination. Prior to challenge, pigs given 2 doses of the vaccine demonstrated boosted levels of antibody compared with those given a single dose, and all surviving pigs had increased neutralization titres over pre-challenge levels. Following challenge, pigs were monitored for clinical signs of disease, with blood and nasal swabs collected for virus isolation. All control animals became sick with elevated temperatures for 6 days post challenge, whereas; vaccinated animals displayed an increase in body temperature for only 2-3 days. Control pigs and those given a single dose all lost condition, but the group given 2 doses remained healthy. At postmortem, gross lesions of pneumonia only occurred in control animals and those given a single dose of vaccine. Histology carried out on the brains of all animals demonstrated a difference in severity of infection and frequency of immunohistochemical antigen detection between test animals, with control and single dose groups being most severely affected and pigs given 2 doses the least. Virus isolation studies demonstrated that no viraemia could be detected, but virus was found in nasal swabs from some animals in both groups of vaccinates following challenge.

Experimental hendra virus infectionin pregnant guinea-pigs and fruit Bats (Pteropus poliocephalus).

Antibodies to Hendra virus (HeV) have been found in a high percentage of fruit bats (Pteropus spp.) in Australia, indicating a possible reservoir for the virus. The aim of the experiments reported here was to investigate transplacental infection as a possible mode of transmission of the virus in fruit bats and other animals. In a first experiment, 18 pregnant guinea-pigs in the mid-stage of gestation were inoculated with HeV, as an experimental model in a conventional laboratory animal. Nine developed HeV disease as confirmed by viral isolation, histopathology and immunohistochemistry. In five of the nine clinically affected guinea-pigs there was necrosis and strong positive immunostaining in the placentas in an indirect immunoperoxidase (IPX) test for HeV antigen. One of these five guinea-pigs aborted and HeV was isolated from its three fetuses, one of which was also positive to the IPX test. In three other sick guinea-pig dams, virus was isolated from fetuses, and there was positive immunostaining in two of the latter. In a second experiment, four fruit bats were inoculated with a similar dose of HeV. (A further four guinea-pigs inoculated at the same time developed severe disease, indicating adequate virulence.) Two bats were killed at 10 days post-inoculation and two were killed at 21 days. In these bats, no overt clinical disease was observed, but subclinical disease occurred, as indicated by viral isolation, seroconversion, vascular lesions and positive immunostaining. Transplacental transmission was indicated by positive immunostaining in two placentas and confirmed by isolation of virus from one of the associated fetuses.

Hendra and Nipah virus infections.

The most important clinical and pathological manifestation of Hendra virus infection in horses and humans is that of severe interstitial pneumonia caused by viral infection of small blood vessels. The virus is also capable of causing nervous disease. Hendra virus is not contagious in horses and is spread by close contact with body fluids, such as froth from infected lungs. Diagnosis should be based on the laboratory examination of blood, lung, kidney, spleen, and, if nervous signs are present, also of the brain. Evidence of infection with the more recently identified and related Nipah virus was found in the brain of one horse in which there was inflammation of the meningeal blood vessels. Fruit bats, especially Pteropus s., have been incriminated as the natural and reservoir hosts of both Hendra and Nipah viruses.

Histopathology and immunohistochemistry of bats infected by Australian bat lyssavirus.

OBJECTIVE: To describe the lesions and distribution of viral antigens in bats infected by Australian bat lyssavirus. DESIGN: A retrospective histopathological and immunohistochemical study of bats naturally infected with the virus. PROCEDURE: Tissues from 37 infected bats were examined. Nineteen flying foxes (fruit bats) and two insectivorous bats were examined in detail. Brains of another 16 flying foxes were poorly fixed and were examined less fully. RESULT: Lesions varied considerably between individuals and, where present, were mostly those of nonsuppurative meningoencephalomyelitis and ganglioneuritis similar to lesions seen in rabies and rabies-like diseases. The number of cells with intracytoplasmic inclusion bodies (Negri bodies) was variable; none were seen in some bats. Intracytoplasmic vacuolation of neurons was a common finding. Lesions occurred throughout the central nervous system but were most frequent and severe in the hippocampus, thalamus and midbrain, and medulla oblongata and pons. Indirect immunoperoxidase tests for lyssavirus antigen reactions varied in intensity and distribution, but also occurred mostly in the hippocampus, thalamus and midbrain, and medulla oblongata and pons. In peripheral tissues, reactions were seen in autonomic ganglia, in nerve plexuses of the gastrointestinal tract, in nervous tissues within muscles and immediately adjacent to individual muscle fibres, in an adrenal medulla, and in epithelial tissues in one of eight salivary glands examined. CONCLUSION: The main lesion in Australian bat lyssavirus infection is nonsuppurative inflammation similar to that seen in rabies and other rabies-like diseases, except that the number of Negri bodies is more variable. Reactions to immunoperoxidase tests for lyssavirus vary in intensity and distribution and may occur in both central and peripheral nervous systems. These reactions do not always occur in the salivary glands, even if brain infection is present.

Epidemic of blindness in kangaroos--evidence of a viral aetiology.

OBJECTIVE: To determine the cause of an epidemic of blindness in kangaroos. DESIGN AND PROCEDURES: Laboratory examinations were made of eyes and brains of a large number of kangaroos using serological, virological, histopathological, electron microscopical, immunohistochemical methods, and PCR with cDNA sequencing. In addition, potential insect viral vectors identified during the disease outbreak were examined for specific viral genomic sequences. SAMPLE POPULATION: For histopathological analysis, 55 apparently blind and 18 apparently normal wild kangaroos and wallabies were obtained from New South Wales, Victoria, South Australia, and Western Australia. A total of 437 wild kangaroos and wallabies (including 23 animals with apparent blindness) were examined serologically. RESULTS: Orbiviruses of the Wallal and Warrego serogroups were isolated from kangaroos affected with blindness in a major epidemic in south-eastern Australia in 1994 and 1995 and extending to Western Australia in 1995/96. Histopathological examinations showed severe degeneration and inflammation in the eyes, and mild inflammation in the brains. In affected retinas, Wallal virus antigen was detected by immunohistochemical analysis and orbiviruses were seen in electron microscopy. There was serological variation in the newly isolated Wallal virus from archival Wallal virus that had been isolated in northern Australia. There were also variations of up to 20% in genotype sequence from the reference archival virus. Polymerase chain reactions showed that Wallal virus was present during the epidemic in three species of midges, Culicoides austropalpalis, C dycei and C marksi. Wallal virus nucleic acid was also detected by PCR in a paraffin-embedded retina taken from a blind kangaroo in 1975. CONCLUSION: Wallal virus and perhaps also Warrego virus are the cause of the outbreak of blindness in kangaroos. Other viruses may also be involved, but the evidence in this paper indicates a variant of Wallal virus, an orbivirus transmitted by midges, has the strongest aetiological association, and immunohistochemical analysis implicates it as the most damaging factor in the affected eyes.

Immunohistochemistry in the identification of a number of new diseases in Australia.

Immunohistochemistry plays an important part in the diagnosis of some viral diseases. Demonstration of viral antigen in a lesion is an important contribution to diagnosis, either at the time of investigation or retrospectively. At the CSIRO Australian Animal Health Laboratory, the most frequent use of immunohistochemistry has been in the diagnosis of the important avian diseases, highly pathogenic avian influenza and Newcastle disease. The technology took key roles in the diagnoses of Hendra virus infections, and, later, an immunoperoxidase test gave the first indication of the existence of Australian bat lyssavirus. The test can often confirm that a virus isolated in an animal is the actual virus causing disease and not a coincidental isolation. Good examples of that in some more new diseases were the association of Wallal virus with blindness in kangaroos, and of the new porcine Menangle virus in natural and experimental cerebral disease in foetal piglets.

Transmission studies of Hendra virus (equine morbillivirus) in fruit bats, horses and cats.

OBJECTIVE: To determine the infectivity and transmissibility of Hendra virus (HeV). DESIGN: A disease transmission study using fruit bats, horses and cats. PROCEDURE: Eight grey-headed fruit bats (Pteropus poliocephalus) were inoculated and housed in contact with three uninfected bats and two uninfected horses. In a second experiment, four horses were inoculated by subcutaneous injection and intranasal inoculation and housed in contact with three uninfected horses and six uninfected cats. In a third experiment, 12 cats were inoculated and housed in contact with three uninfected horses. Two surviving horses were inoculated at the conclusion of the third experiment: the first orally and the second by nasal swabbing. All animals were necropsied and examined by gross and microscopic pathological methods, immunoperoxidase to detect viral antigen in formalin-fixed tissues, virus isolation was attempted on tissues and SNT and ELISA methods were used to detect HeV-specific antibody. RESULTS: Clinical disease was not observed in the fruit bats, although six of eight inoculated bats developed antibody against HeV, and two of six developed vascular lesions which contained viral antigen. The in-contact bats and horses did not seroconvert. Three of four horses that were inoculated developed acute disease, but in-contact horses and cats were not infected. In the third experiment, one of three in-contact horses contracted disease. At the time of necropsy, high titres of HeV were detected in the kidneys of six acutely infected horses, in the urine of four horses and the mouth of two, but not in the nasal cavities or tracheas. CONCLUSIONS: Grey-headed fruit bats seroconvert and develop subclinical disease when inoculated with HeV. Horses can be infected by oronasal routes and can excrete HeV in urine and saliva. It is possible to transmit HeV from cats to horses. Transmission from P poliocephalus to horses could not be proven and neither could transmission from horses to horses or horses to cats. Under the experimental conditions of the study the virus is not highly contagious.

Lesions of experimental equine morbillivirus pneumonia in horses.

Laboratory examinations of equine morbillivirus included experimental reproductions of the disease caused by the virus by transmission of mixed lung and spleen taken from two field equine cases into two horses and by inoculating tissue culture virus into a further two horses. The most distinctive gross lesions of the diseases that developed in three of the horses was that of pulmonary edema characterized by gelatinous distension of subpleural lymphatics. Histologically, the lesions in the lungs were those of serofibrinous alveolar edema, alveolar macrophages, hemorrhage, thrombosis of capillaries, and syncytial cells. Clearly defined vascular lesions in three horses that became clinically affected within 8 days of inoculation of virus included intramural hemorrhage, edema, and necrosis and syncytial cells in the endothelium of pulmonary vessels (approximately 40-70 microm in diameter). Vascular lesions accompanied by parenchymal degeneration were also seen in the heart, kidney, brain, spleen, lymph node, and stomach. A fourth horse, which survived for 12 days, had detectable lesions only in the lungs, which were more chronic than those in the other three horses, a greater degree of cellular infiltration, and fewer well-defined vascular lesions. Sections stained by an indirect immunocytochemical method showed equine morbillivirus antigen was present in the vascular lesions and along alveolar walls. When endothelial cells were examined by electron microscope, cytoplasmic virus inclusion bodies containing filamentous structures were seen that reacted to an immunogold test to equine morbillivirus antigen. The presence of the syncytia in the small blood vessels in the lungs and other organs was interpreted as an important characteristic of the disease and consistent with a reaction to a morbillivirus.

The lesions of experimental equine morbillivirus disease in cats and guinea pigs.

Nine cats and four guinea pigs became affected with severe disease during experiments on the infectivity of equine morbillivirus, a newly recognized cause of respiratory disease in horses and humans. Four of the cats were challenged by subcutaneous inoculation, two by intranasal installation, two by oral dosage, and one by direct contact with a cat previously infected by subcutaneous inoculation. All four guinea pigs were inoculated subcutaneously. Gross pathology seen in all affected cats was characterized by hydrothorax and dark, heavy, wet, congested and/or hemorrhagic lungs with froth sometimes found in the respiratory passages. Pulmonary lymph nodes were enlarged and edematous. Six cats also had congested ceca with accompanying edema of mesenteric lymph nodes. Histologically, the lesions in the lungs of the cats were those of severe interstitial pneumonia characterized by serofibrinous alveolar edema, alveolar macrophages, intra-alveolar hemorrhage, thrombosis of small veins, alveolar wall necrosis, and syncytial cells. Clearly defined vascular lesions included intramural hemorrhage, edema, and necrosis and syncytial cells in the endothelium of pulmonary arteries and veins, 20-80 microm in diameter. Vascular lesions accompanied by parenchymal degeneration were also seen in the gastrointestinal and lymphoid organs. Syncytial cells were also visible in the lymphoid tissues of lymph nodes, spleen, and Peyer's patches. At necropsy, all guinea pigs were cyanosed and had congestion and edema in the gastrointestinal tract. Histologically, there was widespread vascular disease in arteries and veins, 20-80 microm in diameter, in many organs such as the lungs, kidneys, spleens, lymph nodes, gastrointestinal tracts, and skeletal and intercostal muscles, but there was no severe pulmonary edema as seen in horses and cats. Sections of tissues of the cats and guinea pigs, examined by indirect immunocytochemical stains, confirmed that the vascular damage was associated with the presence of equine morbillivirus antigen. The syncytia in small blood vessels in the lungs and other organs of both cats and guinea pigs were similar to those seen in horses, and their presence was interpreted as an important characteristic of the disease consistent with a reaction to a morbillivirus.

Recombinant fowlpox virus vaccines against Australian virulent Marek's disease virus: gene sequence analysis and comparison of vaccine efficacy in specific pathogen free and production chickens.

We have cloned and sequenced the glycoprotein genes gB, gC and gD of the Australian virulent Marek's disease virus (MDV) isolate Woodlands No. 1. The glycoprotein gB and gC sequences were identical to the homologs of other virulent MDV type 1 strains, and the glycoprotein gD sequence contained only one unique amino acid substitution. Recombinant fowlpox viruses (rFPVs) expressing the MDV glycoprotein genes were constructed and their efficacy as vaccines was evaluated in specific pathogen free (SPF) and production chickens. Vaccination with the FPV-gB recombinant protected SPF chickens from Marek's disease mortality and tumour formation following challenge with virulent MDV Woodlands No. 1. The degree of protection from Marek's disease was dependent on the vaccine dose and route of inoculation. The rFPVs expressing gC or gD did not provide protection from Marek's disease. A rFPV expressing both gB and gC did not provide enhanced protection in comparison with the rFPV-gB alone. The rFPV-gB vaccine failed to protect commercial chickens from MD mortality and provided little protection from tumour formation in comparison with the commercial herpesvirus of turkey (HVT) vaccine. The failure to provide protection against MD may be related to the impact of maternally derived immunity to MDV and FPV and possibly the genotype of the chickens.

Encephalitis caused by a Lyssavirus in fruit bats in Australia.

This report describes the first pathologic and immunohistochemical recognition in Australia of a rabies-like disease in a native mammal, a fruit bat, the black flying fox (Pteropus alecto). A virus with close serologic and genetic relationships to members of the Lyssavirus genus of the family Rhabdoviridae was isolated in mice from the tissue homogenates of a sick juvenile animal.