Epizootic haemorrhagic disease.

Summary Epizootic haemorrhagic disease (EHD) is an arthropod-transmitted viral disease of certain wild ungulates, notably North American white-tailed deer and, more rarely, cattle. The disease in white-tailed deer results from vascular injury analogous to that caused by bluetongue virus (BTV), to which EHD virus (EHDV) is closely related. There are seven serotypes of EHDV recognised, and Ibaraki virus, which is the cause of sporadic disease outbreaks in cattle in Asia, is included in EHDV serotype 2. The global distribution and epidemiology of BTV and EHDV infections are also similar, as both viruses occur throughout temperate and tropical regions of the world where they are transmitted by biting Culicoides midges and infect a wide variety of domestic and wild ungulates. However, the global distribution and epidemiology of EHDV infection are less well characterised than they are for BTV. Whereas most natural and experimental EHDV infections (other than Ibaraki virus infection) of livestock are subclinical or asymptomatic, outbreaks of EHD have recently been reported among cattle in the Mediterranean Basin, Reunion Island, South Africa, and the United States. Accurate and convenient laboratory tests are increasingly available for the sensitive and specific serological and virological diagnosis of EHDV infection and confirmation of EHD in animals, but commercial vaccines are available only for prevention of Ibaraki disease and not for protection against other strains and serotypes of EHDV.

Bluetongue.

Summary Bluetongue (BT) is an arthropod-transmitted viral disease of non-African ungulates, principally sheep. The disease results from vascular injury analogous to that of human haemorrhagic viral fevers, with characteristic tissue infarction, haemorrhage, vascular leakage, oedema, and hypovolaemic shock. Importantly, BT is not zoonotic. Bluetongue virus (BTV) infection of ruminants and vector Culicoides midges is endemic throughout many tropical and temperate regions of the world; however, within this global range the virus exists within relatively discrete ecosystems (syn. episystems) where specific constellations of BTV serotypes are spread by different species of biting Culicoides midges. Recently discovered goat-associated BTVs, notably BTV serotype 25 (BTV-25) in central Europe, appear to have distinctive biological properties and an epidemiology that is not reliant on Culicoides midges as vectors for virus transmission. Bluetongue virus infection of ruminants is often subclinical, but outbreaks of severe disease occur regularly at the upper and lower limits of the virus's global range, where infection is distinctly seasonal. There have been recent regional alterations in the global distribution of BTV infection, particularly in Europe. It is proposed that climate change is responsible for these events through its impact on vector midges. However, the role of anthropogenic factors in mediating emergence of BTV into new areas remains poorly defined; for example, it is not clear to what extent anthropogenic factors were responsible for the recent translocation to northern and eastern Europe of live attenuated vaccine viruses and an especially virulent strain of BTV-8 with distinctive properties. Without thorough characterisation of all environmental and anthropogenic drivers of the recent emergence of BT in northern Europe and elsewhere, it is difficult to predict what the future holds in terms of global emergence of BTV infection. Accurate and convenient laboratory tests are available for the sensitive and specific serological and virological diagnosis of BTV infection and confirmation of BT in animals. Prevention and control strategies for BT are largely reactive in nature, and typically are reliant on vaccination of susceptible livestock and restrictions on animal trade and movement.

Infection and disease in reservoir and spillover hosts: determinants of pathogen emergence.

Infection and disease in reservoir and spillover hosts determine patterns of infectious agent availability and opportunities for infection, which then govern the process of transmission between susceptible species. In this chapter, using the zoonotic agents Hendra virus and Nipah virus as examples, the pathogenesis of infection in various species including the wildlife reservoirs and domestic spillover hosts is reviewed with an emphasis on the aspects of pathogenesis which contribute to the dissemination of infection. Through these discussions, the emergence of these zoonotic agents is explored.

Experimental Nipah virus infection in pteropid bats (Pteropus poliocephalus).

Seventeen grey-headed fruit bats (Pteropus poliocephalus) were inoculated subcutaneously with an isolate of Nipah virus derived from a fatally infected human. A control group of eight guinea-pigs was inoculated intraperitoneally with the same isolate in order to confirm virulence. Three of eight infected guinea-pigs developed clinical signs 7-9 days post-inoculation. Infected fruit bats developed a subclinical infection characterized by the transient presence of virus within selected viscera, episodic viral excretion and seroconversion. A range of histopathological changes was observed within the tissues of infected bats. Nipah virus was excreted in bat urine while neutralizing antibody was present in serum. This intermittent, low-level excretion of Nipah virus in the urine of bats may be sufficient to sustain the net reproductive value of the virus in a species where there is regular urine contamination of the fur, mutual grooming, and where urine droplets are a feature of the environment.

Japanese encephalitis in a racing thoroughbred gelding in Hong Kong.

A horse in Hong Kong that had been vaccinated against Japanese encephalitis suffered a pyrexic episode that culminated in a hyperexcitable state and self-inflicted trauma. Japanese encephalitis was diagnosed on the basis of clinical, pathological and serological observations, and confirmed by the detection of genomic sequences of the virus in spinal cord tissue. Phylogenetic analyses of E gene and NS5-3'UTR sequences revealed divergent clustering of these segments with previously described genotypes, suggesting the possibility that the horse might have been infected with a recombinant between genotype I and genotype II viruses. Horses are considered to be dead-end hosts for the disease, but the occurrence of an infected horse in a population may have implications for the health status of the national herd. The effect that this case had on the horse industry in Hong Kong is discussed with specific reference to the movement of horses and the vaccination programme for Japanese encephalitis.

Genetic diversity of bluetongue viruses in south east Asia.

Bluetongue viruses (BTV) were isolated from sentinel cattle in Malaysia and at two sites in Indonesia. We identified eight serotypes some of which appeared to have a wide distribution throughout this region, while others were only isolated in Malaysia or Australia. Nearly half of the 24 known BTV serotypes have now been identified in Asia. Further, we investigated the genetic diversity of their RNA segments 3 and 10. Using partial nucleotide sequences of the RNA segment 3 (540 bp) which codes for the conserved core protein (VP3), the BTV isolates were found to be unique to the previously defined Australasian topotype and could be further subdivided into four distinct clades or genotypes. Certain of these genotypes appeared to be geographically restricted while others were distributed widely throughout the region. Similarly, the complete nucleotide sequences of the RNA segment 10 (822 bp), coding for the non-structural protein (NS3/3A), were also conserved and grouped into the five genotypes; the BTV isolates could be grouped into three Asian genotypes and two Nth American/Sth African genotypes.

Experimental infections of pigs with Japanese encephalitis virus and closely related Australian flaviviruses.

The flavivirus Japanese encephalitis (JE) virus has recently emerged in the Australasian region. To investigate the involvement of infections with related enzootic flaviviruses, namely Murray Valley encephalitis (MVE) virus and Kunjin (KUN) virus, on immunity of pigs to JE virus and to provide a basis for interpretation of serologic data, experimental infections were conducted with combinations of these viruses. Antibody responses to primary and secondary infections were evaluated using panels of monoclonal antibody-based blocking enzyme-linked immunosorbent assays and microtiter serum neutralization tests (mSNTs). Identification of the primary infecting virus was possible only using the mSNTs. Following challenge, unequivocal diagnosis was impossible due to variation in immune responses between animals and broadened and/or anamnestic responses. Viremia for JE virus was readily detected in pigs following primary infection, but was not detected following prior exposure to MVE or KUN viruses. Boosted levels of existing cross-neutralizing antibodies to JE virus suggested a role for this response in suppressing JE viremia.

Isolation of bluetongue virus serotype 21 from Culicoides spp. in Indonesia.

The isolation of a bluetongue (BLU) virus from Culicoides spp. in Indonesia is reported. BLU serotype 21 was isolated from a mixed pool of C. fulvus and C. orientalis of the Avaritia subgenus.

Australian-Indonesian collaboration in veterinary arbovirology--a review.

Australian-Indonesian collaboration in veterinary development programs has led to significant advances in the study of arboviruses. This paper reviews the resulting knowledge of arboviral infections of livestock in Indonesia. The first recognized arboviral disease of animals in Indonesia was bovine ephemeral fever. Serology indicates that the virus is widespread, as are related rhabdoviruses. Local sheep appear resistant to bluetongue disease, but imported sheep have suffered mortalities. Bluetongue viral serotypes 1, 7, 9, 12, 21 and 23 have been isolated from sentinel cattle; 1, 21 and 23 at widely separate locations. Bluetongue serotype 21 has been isolated from Culicoides spp. Serological reactors to Akabane virus are widespread, as are reactors to the flavivirus group. Japanese encephalitis, isolated from sentinel pigs, is the flavivirus of most veterinary importance but the limit of its easterly distribution is unknown. Many of the arboviruses present in Indonesia are also present in Australia and elsewhere in Asia. Their patterns of mobility among countries in the region are largely undescribed, but there are opportunities for further regional collaboration.

Development of a polymerase chain reaction to detect Vietnamese isolates of duck virus enteritis.

A polymerase chain reaction (PCR) method for the detection of duck virus enteritis (DVE) virus in tissues of infected and affected ducks, and in cell culture was developed. This required us to obtain specific nucleotide sequence information as we could not find any specific data about the genome of the virus. We found the assay to be highly effective in detecting the virus under experimental conditions and to be easily transferred to laboratories in Vietnam where it is being used in studies on the epidemiology of the disease. We have applied this simple and rapid diagnostic method to the detection of DVE isolates grown in cell culture and tissues from infected birds. The assay was also able to differentiate DVE from other avian herpesviruses, such as Marek's disease, infectious laryngotracheitis virus and goose herpesvirus.

Antibodies against certain bluetongue and epizootic haemorrhagic disease viral serotypes in Indonesian ruminants.

The orbiviruses contain several important viruses of livestock including bluetongue (BT) and epizootic haemorrhagic disease of deer (EHD) which share some group antigens. Preliminary screening of sera for antibodies to orbiviruses by the agar gel immunodiffusion (AGID) test has previously revealed widespread infections with the BT group in Indonesia. However serum neutralization (SN) tests give a more accurate estimate of exposure to each serotype in the BT and EHD groups, and in this study were applied to sera that had reacted previously in the AGID test. Five different serotypes of BT and one serotype of EHD virus were studied. Reactors to BT serotype 20 were the most prevalent, followed by EHD type 5 and BT types 21, 12, 1 and 17. Antibodies against BT serotype 20 were present in cattle, buffaloes, goats and sheep, but were most common in buffaloes. Buffaloes showed the highest exposure to the BT serotypes tested. Antibody to EHD type 5 occurred most frequently in cattle. Antibodies against all BT and EHD serotypes tested were found in buffaloes and cattle while goats had antibodies against BT types 20, 21 and EHD type 5 and sheep had antibodies only against BT type 20.

An outbreak of highly pathogenic avian influenza in Australia in 1997 caused by an H7N4 virus.

In November of 1997 an outbreak of highly pathogenic avian influenza occurred near the town of Tamworth, in northern New South Wales, Australia. The viruses isolated from chickens on two commercial chicken farms were identified as H7N4 viruses, with hemagglutinin cleavage site amino acid sequences of RKRKRG and intravenous pathogenicity indices of 2.52 and 2.90, respectively. A virus with an identical nucleotide sequence, but with an intravenous pathogenicity index of 1.30, was also isolated from cloacal swabs collected from asymptomatic emus kept on a third property.

Non-effect of regional lymph node removal on growth of ovine aural squamous cell carcinoma.

The effect of ipsilateral parotid lymphadenectomy on growth of lesions of aural squamous cell carcinoma in 10 adult ewes was compared over a 6-month period, with matched, tumour-affected controls that were not subjected to parotid lymphadenectomy. There was no significant difference between groups, indicating that the regional lymph node had no influence on the growth of the tumour.

A study of immunoglobulin-containing and other cells in ovine aural squamous cell carcinoma.

The peroxidase-antiperoxidase method was used to quantify immunoglobulin (Ig)-containing cells at the host-tumour interface in ovine aural squamous cell carcinoma lesions at various stages of development. IgM-containing cells were never observed; only scattered, but strongly staining, IgA-containing cells were present. IgG-containing cells predominated; their prevalence increased from 0.17 +/- 0.34 cells per high power field (hpf; mean +/- standard deviation) in precursor lesions to a maximum of 13.6 +/- 2.6 cells per hpf in overt tumours which had not metastasized. Their prevalence in overt tumours which had metastasized was 4.6 +/- 3.9 cells per hpf. Quantitation of pyroninophils in sections stained by the methyl green-pyronin method showed a greater prevalence of plasma cells than IgG-containing cells, but a similar pattern of change was apparent. Mast cells were most prevalent in early stages, but no significant change in eosinophil numbers was observed.

Diseases in young farmed crocodiles in Irian Jaya.

Thirty-eight young crocodiles that were emaciated and were euthanased or were found dead on 12 farms in Irian Jaya were examined post mortem. Major diseases were coccidiosis (nine crocodiles), pentastomiasis (four), visceral gout (two) and bacterial pneumonia and septicaemia (two). Other diseases and infections were steatitis, fungal pneumonia, gastric capillariasis, haemogregarine infection, ascariasis, filarioid infection and the presence of flukes in the intestine, kidney and blood. Multiple parasitism due to the collection of hatchlings in the wild was considered the primary cause of the ill-thrift and death of the crocodiles.

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.

Serological survey for Ehrlichia canis in urban dogs from the major population centres of northern Australia.

OBJECTIVE: To detect evidence of Ehrlichia canis infection of dogs from the major population centres of northern Australia, if present. DESIGN: Serological investigation for E. canis. PROCEDURE: The sera of 316 domestic dogs, collected from the northern Australian population centres of Townsville, Cairns, Darwin, Kununurra and Broome from May 1997 to August 1999, were investigated for evidence of infection with E. canis. Samples were tested for antibodies to E. canis using an indirect fluorescent antibody (IFA) test. The buffy coats from blood of dogs whose serum reacted in the IFA test were subsequently tested with a nested PCR to detect E. canis DNA. When available, blood from these dogs was injected into suckling mice, which were then examined for clinical disease and tested for the presence of E. canis antibodies. RESULTS: Of the 316 samples tested seven reacted in the IFA test for E. canis. None of the dogs from which these samples were obtained exhibited clinical signs of acute or chronic ehrlichiosis. The six positive samples available for testing were negative when tested with the nested PCR. Suckling mice inoculated with blood from three of the dogs whose serum was positive by IFA test showed no signs of clinical disease nor did their give positive reactions in the IFA test. CONCLUSIONS: No evidence of E. canis infection was confirmed in any of the dogs examined. Northern Australia would appear to remain free of this obligate parasite.

Reproductive disease and congenital malformations caused by Menangle virus in pigs.

OBJECTIVE: To describe a new syndrome characterised by embryonic mortalities, stillbirths, mummified foetuses and congenital malformations in a herd of intensively farmed pigs. DESIGN: Field observations, laboratory investigations and examination of breeding records. PROCEDURE: Pathology examinations were performed on mummified and congenitally deformed piglets during an outbreak of reproductive disease at a 2600 sow intensive piggery in New South Wales from April to October 1997. Reproductive performance was monitored during the outbreak and breeding records were examined retrospectively. Serum and tissue samples from pigs were tested for evidence of infection with known porcine pathogens and for a new virus, Menangle virus, isolated from stillborn piglets with deformities from the affected piggery in August 1997. RESULTS: Reproductive disease occurred sequentially in all four breeding units at the affected piggery over a period of 21 weeks. The farrowing percentages in each unit decreased from 80 to 82% before the outbreak to 63 to 78% during the outbreak and the number of live piglets per litter declined from a mean of 9.6 to 9.8 before the outbreak to 7.2 to 8.9 during the outbreak. The proportion of affected litters (litters with less than six liveborn piglets) was highest (64%) in the sixth week of the outbreak. Mummified foetuses, stillborn piglets with arthrogryposis, craniofacial deformities and degeneration of the brain and spinal cord, were observed along with occasional abortions. Sera from sows that produced affected litters contained neutralising antibodies against Menangle virus and there was evidence that this virus had been introduced to the piggery in February 1997. CONCLUSIONS: Reproductive disease in pigs due to Menangle virus was characterised by stillbirths, mummification, embryonic death and infertility, along with abortions, skeletal deformities and degeneration of the brain and spinal cord in affected foetuses and stillborn piglets.