Foot and mouth disease in the Lao People's Democratic Republic: I. A review of recent outbreaks and lessons from control programmes.

Foot and mouth disease (FMD) causes sporadic disease outbreaks in the Lao People's Democratic Republic (Lao PDR). As the Lao PDR is a major thoroughfare for transboundary animal movements, regular FMD outbreaks occur, causing economic hardship for farmers and their families. In this review of the recent history of FMD in the Lao PDR between 1997 and 2006, the authors examine the virological and epidemiological aspects of the disease and appropriate control measures, including the distribution of outbreaks, causative serotypes and the molecular epidemiology of the viruses, as well as large-scale vaccination programmes. The dominant serotype, type O, was reported every year from 1998 to 2005. The majority of outbreaks occurred in Vientiane Capital (n = 42; 28%) and the highest number of outbreaks were reported in cattle (n = 94; 61%); followed by buffalo (n = 41; 27%) and pigs (n = 18; 12%). All type A outbreaks occurred in cattle. Type Asia 1 outbreaks were reported in the central provinces around Vientiane Capital between 1996 and 1998.

Foot and mouth disease in the Lao People's Democratic Republic: II. Seroprevalence estimates, using structured surveillance and surveys of abattoirs.

An examination of the seroprevalence of foot and mouth disease (FMD) virus was conducted in the Lao People's Democratic Republic (Lao PDR) from 1996 to 2005, using structured surveillance and abattoir-based studies. Under structured surveillance, seropositivity ranged from 65.7% (Vientiane Capital, 1996) to 3% (Houaphan, 2005) for cattle and buffalo; and from 2.8% (Vientiane Capital, 1998) to 0% in separate studies of pigs. In each study, species composition was significantly associated with seroprevalence rates. For abattoir surveys, the majority of samples (60.5%) came from Vientiane Capital (33.0%), Savannakhet (14.0%) and Champasak (13.5%) provinces. The overall proportion of animals testing positive for the presence of antibodies against the FMD virus was 18.7% (ranging from 50.8% in Vientiane Province to 1% in Phongsali). Generally, antibodies against serotype O were the most prevalent. Cattle and buffalo that tested as seropositive were significantly older than the seronegative animals (p < 0.00005). The overall proportional seropositivity was significantly different for different species, as was the case with the antibodies against serotypes O, A and Asia 1. Some 22% of cattle, 55% of buffalo and 23% of pigs demonstrated seropositivity but this varied significantly between provinces.

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.

Comparative susceptibility of indigenous and improved pig breeds to Classical swine fever virus infection: practical and epidemiological implications in a subsistence-based, developing country setting.

This study investigated the comparative susceptibility of indigenous Moo Laat and improved Large White/Landrace pig breeds to infection with classical swine fever virus (CSFV) under controlled conditions in the Lao People's Democratic Republic (Lao PDR). The Moo Laat (ML) and Large White/Landrace cross-breed (LWC) pigs were inoculated with a standard challenge strain designated Lao/Kham225 (infectivity titre of 10(2.75) TCID50/ml). The results demonstrated that both the native breed and an improved pig breed are fully susceptible to CSFV infection and the mortality rate is high. LWC pigs demonstrated lower (or shorter) survival times (50% survival time: 11 days), earlier and higher pyrexia and earlier onset of viraemia compared to ML pigs (50% survival time: 18 days). In the context of village-based pig production, the longer time from infection to death in native ML pigs means that incubating or early sick pigs are likely to be sold once an outbreak of CSF is recognized in a village. This increased longevity probably contributes to the maintenance and spread of disease in a population where generally the contact rate is low.

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.

Pathogenesis studies with Australian bat lyssavirus in grey-headed flying foxes (Pteropus poliocephalus).

OBJECTIVE: To examine the susceptibility of the grey-headed flying fox (Pteropus poliocephalus) to Australian bat lyssavirus (ABL), and to provide preliminary observations on the pathogenesis of the disease in flying foxes. PROCEDURE: Ten flying foxes were inoculated intramuscularly with ABL, and four with a bat-associated rabies virus. Inoculated animals were observed daily, and clinical samples collected every 9 to 14 days. Animals with abnormal clinical signs were euthanased, and samples collected for histological, serological, virological and immunohistological examinations. At 3 months post inoculation (PI), all survivors were euthanased, and each submitted to a similar examination. RESULTS: Three ABL-inoculated flying foxes, and two rabies-inoculated animals developed abnormal clinical signs between 15 and 24 days PI. All three ABL-inoculated animals had histological lesions consistent with a lyssavirus infection, and lyssaviral antigen was identified in the central nervous system (CNS) of each. Virus was isolated from the brain of two affected animals. Of the rabies-inoculated flying foxes, both had histological lesions and viral antigen in the CNS. Virus was recovered from the brain of only one. None of the five affected flying foxes developed anti-lyssavirus antibodies, but, by 3 months PI, five of the seven ABL-inoculated survivors, and one of the two rabies virus-inoculated survivors, had seroconverted. The dynamics of the immune responses were quite variable. CONCLUSIONS: The response of flying foxes to ABL, administered by a peripheral route of inoculation, was similar to that of bats inoculated peripherally with bat-derived rabies viruses.

Experimental Nipah virus infection in pigs and cats.

A human isolate of Nipah virus from an outbreak of febrile encephalitis in Malaysia that coincided with a field outbreak of disease in pigs was used to infect eight 6-week-old pigs orally or subcutaneously and two cats oronasally. In pigs, the virus induced a respiratory and neurological syndrome consistent with that observed in the Malaysian pigs. Not all the pigs showed clinical signs, but Nipah virus was recovered from the nose and oropharynx of both clinically and sub-clinically infected animals. Natural infection of in-contact pigs, which was readily demonstrated, appeared to be acute and self-limiting. Subclinical infections occurred in both inoculated and in-contact pigs. Respiratory and neurological disease was also produced in the cats, with recovery of virus from urine as well as from the oropharynx. The clinical and pathological syndrome induced by Nipah virus in cats was comparable with that associated with Hendra virus infection in this species, except that in fatal infection with Nipah virus there was extensive inflammation of the respiratory epithelium, associated with the presence of viral antigen. Viral shedding via the nasopharynx, as observed in pigs and cats in the present study, was not a regular feature of earlier reports of experimental Hendra virus infection in cats and horses. The findings indicate the possibility of field transmission of Nipah virus between pigs via respiratory and oropharyngeal secretions.

Evidence for insect transmission of rabbit haemorrhagic disease virus.

The spread of rabbit haemorrhagic disease (RHD) virus from quarantine on Wardang Island to mainland Australia in 1995 suggested that insects could be potential vectors. Field observations and laboratory experiments were conducted to address aspects of this hypothesis. Firstly, the variation in insect populations on the island during the field trials was examined. There was approximately a 1,000-fold increase in the number of bushflies, Musca vetustissima, shortly before the spread of the virus. Secondly, M. vetustissima were tested in the laboratory as potential vectors of RHD virus, and it was demonstrated that disease could be transmitted between rabbits by flies. Finally, 13 of 16 insect samples, collected from Wardang Island and from several sites on the mainland following the spread of virus off the island, were positive for the presence of RHD virus by a specific polymerase chain reaction (PCR). Only one sample contained sufficient infectious virus to kill a susceptible rabbit. These data, combined with previously published information on fly biology, suggested that flies, particularly bushflies, may be involved in the transmission of RHD virus. Other possible routes of spread were not assessed in this study.

Serological examination for evidence of infection with Hendra and Nipah viruses in Queensland piggeries.

OBJECTIVE: To examine piggeries in Queensland for evidence of infection with Hendra virus and Nipah virus. DESIGN: A serological survey was designed to provide 99% confidence of detecting at least one infected pig herd in Queensland, assuming that for each virus, at least 5% of herds would have been exposed to virus and that at least 40% of the finisher pigs in these herds would have detectable antibodies to virus. PROCEDURE: A two stage sampling regimen was used. All samples were tested with serum neutralisation tests developed and performed at the Australian Animal Health Laboratory. RESULTS: There was no evidence of antibody to either virus in the 500 samples collected from 100 herds. CONCLUSION: The results of the survey support a case that commercial pigs in Queensland are free of both Hendra virus and Nipah virus infections.

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.

Virulent Newcastle disease in Australia: molecular epidemiological analysis of viruses isolated prior to and during the outbreaks of 1998-2000.

Gene sequence analysis of fusion (F) gene cleavage motifs and haemagglutinin-neuraminidase (HN) carboxyl-terminal extension sequences was used to analyse Newcastle disease viruses (NDV) associated with virulent outbreaks of the disease which occurred in New South Wales, Australia in 1998-2000. PCR fragments were amplified directly from diseased tissue or allantoic fluids and sequence analyses used for phylogenetic comparisons between these viruses and Australian reference NDV. F and HN gene sequence comparison showed a strong relationship to sequences derived from endemic Australian NDV rather than those of overseas viruses or wild bird isolates. Prior to notification of the 1998 outbreak, an NDV was isolated from chickens suffering respiratory disease that appeared to be the progenitor virus from which the virulent virus originated. In turn, these viruses are closely related to two previously isolated 'ancestor' viruses that have the same unique HN extension sequence.

The presence of cross-reactive antibodies to rabbit haemorrhagic disease virus in Australian wild rabbits prior to the escape of virus from quarantine.

Sera collected from Australian wild rabbits prior to the escape of rabbit haemorrhagic disease virus (RHDV) from Wardang Island were examined for RHDV antibodies using purified recombinant capsid protein VP60 expressed from baculovirus. A VP60-based indirect ELISA showed that 196 of 392 wild rabbit sera reacted (OD(450) >0.15) with VP60. Twenty sera (OD(450) ranging from 0.15-2.47), randomly chosen from the 196 positive sera, recognized the 64 kDa VP60 in Western blot analysis, indicating that the reactivity detected in ELISA is indeed specific to the VP60 antigen. In a separate study, sera of 23 rabbits from an RHD-free area after the escape of RHDV were tested by ELISA and 21 of the 23 rabbits were found to be positive. When these rabbits were challenged with a lethal dose of RHDV, 11 out of the 23 rabbits survived. The presence of RHDV-protective antibodies in some of these rabbits suggested that they had been exposed to a pre-existing non-virulent rabbit calicivirus closely related to RHDV. These results highlight the need to study the prevalence of, and to characterize, this viral agent in order to effectively control rabbit populations in Australia and New Zealand.

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 virus disease in horses.

The author provides an account of the discovery of a previously undescribed disease of horses and a description of the studies involved in determining the aetiology of the disease. The causative virus, now named Hendra virus (HeV), is the reference virus for a proposed new genus within the virus family Paramyxoviridae. The virus is a lethal zoonotic agent able to cause natural disease in humans and horses and experimentally induced disease in cats, guinea-pigs and mice. The virus also naturally infects species of the family Megachiroptera, mainly subclinically, and such animals are the natural host of HeV. The virus appears to transmit readily between species of Megachiroptera, but not readily between horses under natural and experimental conditions, or from horses to humans. The method of transmission from bats to horses is not known. Three incidents of HeV disease in horses have been recorded in Australia--two in 1994 which caused the death of two humans and fifteen horses and one in 1999 which involved the death of a single horse. Hendra virus is related to Nipah virus, the virus that caused disease and mortality in humans, pigs, dogs and cats in Malaysia during 1998 and 1999.

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.

Viral haemorrhagic disease of rabbits and human health.

Viral haemorrhagic disease of rabbits (VHD), a potential biological control for wild rabbits in Australia and New Zealand, escaped from quarantined field trials on Wardang Island and spread to the mainland of Australia in October 1995. This study looked for any evidence of infection or illness in people occupationally exposed to the virus. Two hundred and sixty-nine people were interviewed and 259 blood samples were collected. Exposures to VHD-infected rabbits ranged from nil to very high. No VHD antibodies were detected in any of the 259 sera when tested by VHD competitive enzyme immunoassay, which had been validated with 1013 VHDV-specific antibody negative sera. A questionnaire designed to elicit symptoms of disease in a range of organ systems found no significant differences between illness in those exposed and those not exposed to VHD, nor could an association be found between exposure and subsequent episodes of illness. The findings are consistent with the view that exposure to VHD is not associated with infection or disease in humans.

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.