Neuro-angiostrongylosis in wild Black and Grey-headed flying foxes (Pteropus spp)

OBJECTIVE: To identify nematodes seen in histological sections of brains of flying foxes (fruit bats) and describe the associated clinical disease and pathology. PROCEDURES: Gross and histological examination of brains from 86 free-living flying foxes with neurological disease was done as part of an ongoing surveillance program for Australian bat lyssavirus. Worms were recovered, or if seen in histological sections, extracted by maceration of half the brain and identified by microscopic examination. Histological archives were also reviewed. RESULTS: There was histological evidence of angiostrongylosis in 16 of 86 recently submitted flying foxes with neurological disease and in one archival case from 1992. In 10 flying foxes, worms were definitively identified as Angiostrongylus cantonensis fifth-stage larvae. A worm fragment and third stage larvae were identified as Angiostrongylus sp, presumably A cantonensis, in a further three cases. The clinical picture was dominated by paresis, particularly of the hindlimbs, and depression, with flying foxes surviving up to 22 days in the care of wildlife volunteers. Brains containing fifth-stage larvae showed a moderate to severe eosinophilic and granulomatous meningoencephalitis (n = 14), whereas there was virtually no inflammation of the brains of bats which died when infected with only smaller, third-stage larvae (n = 3). There was no histological evidence of pulmonary involvement. CONCLUSION: This is the first report of the recovery and identification of A cantonensis from free-living Australian wildlife. While angiostrongylosis is a common cause of paresis in flying foxes, the initial clinical course cannot be differentiated from Australian bat lyssavirus infection, and wildlife carers should be urged not to attempt to rehabilitate flying foxes with neurological disease.     

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.     

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.     

An unprecedented cluster of Australian bat lyssavirus in Pteropus conspicillatus indicates pre‐flight flying fox pups are at risk of mass infection

In November 2017, two groups of P. conspicillatus pups from separate locations in Far North Queensland presented with neurological signs consistent with Australian bat lyssavirus (ABLV) infection. These pups (n = 11) died over an 11‐day period and were submitted to a government laboratory for testing where ABLV infection was confirmed. Over the next several weeks, additional ABLV cases in flying foxes in Queensland were also detected. Brain tissue from ABLV‐infected flying foxes during this period, as well as archived brain tissue, was selected for next‐generation sequencing. Phylogenetic analysis suggests that the two groups of pups were each infected from single sources. They were likely exposed while in crèche at night as their dams foraged. This study identifies crèche‐age pups at a potentially heightened risk for mass ABLV infection.     

Newly discovered viruses of flying foxes.

Flying foxes have been the focus of research into three newly described viruses from the order Mononegavirales, namely Hendra virus (HeV), Menangle virus and Australian Bat Lyssavirus (ABL). Early investigations indicate that flying foxes are the reservoir host for these viruses. In 1994, two outbreaks of a new zoonotic disease affecting horses and humans occurred in Queensland. The virus which was found to be responsible was called equine morbillivirus (EMV) and has since been renamed HeV. Investigation into the reservoir of HeV has produced evidence that antibodies capable of neutralising HeV have only been detected in flying foxes. Over 20% of flying foxes in eastern Australia have been identified as being seropositive. Additionally six species of flying foxes in Papua New Guinea have tested positive for antibodies to HeV. In 1996 a virus from the family Paramyxoviridae was isolated from the uterine fluid of a female flying fox. Sequencing of 10000 of the 18000 base pairs (bp) has shown that the sequence is identical to the HeV sequence. As part of investigations into HeV, a virus was isolated from a juvenile flying fox which presented with neurological signs in 1996. This virus was characterised as belonging to the family Rhabdoviridae, and was named ABL. Since then four flying fox species and one insectivorous species have tested positive for ABL. The third virus to be detected in flying foxes is Menangle virus, belonging to the family Paramyxoviridae. This virus was responsible for a zoonotic disease affecting pigs and humans in New South Wales in 1997. Antibodies capable of neutralising Menangle virus, were detected in flying foxes.     

Radiographic evaluation of cardiac size in flying fox species (Pteropus rodricensis, P. hypomelanus, and P. vampyrus).

Dilated cardiomyopathy is a relatively common pathology in captive flying foxes (Pteropus spp.). The goal of this study was to establish quantitative reference range measurements that could be used to support a diagnosis of cardiac disease in these animals. Lateral and ventrodorsal thoracic radiographs from apparently healthy flying foxes (n = 66) of three species (Rodriguez island flying fox, P. rodricensis, n = 18; small island flying fox, P. hypomelanus, n = 16; and Malaysian flying fox, P. vampyrus, n = 32) were evaluated objectively to describe the cardiac appearance. Absolute and relative cardiac dimensions also were measured. The same methods were used to evaluate radiographs from flying foxes (n = 9) with known dilated or acute cardiomyopathy. The following ratios were most appropriate for categorizing normal cardiac silhouette size. In the ventrodorsal projection, heart width to thoracic width and heart width to clavicle length were the preferred measurements. In the lateral projection, heart width compared with thoracic height was the preferred measurement. From radiographs of the bats with known dilated and acute cardiomyopathy, the apicobasilar heart length compared with thoracic height and heart width compared with thoracic height on lateral films were the most sensitive ratios for diagnosing cardiomegaly.     

A Pasteurella-like bacterium associated with pneumonia in captive megachiropterans.

A novel Pasteurella-like organism was recovered postmortem from lung tissue of two captive Wahlberg's epauleted fruit bats (Epomophorus wahlbergi), with severe, unilateral pneumonia. The bats had been recently shipped and died shortly after release from a 30-day quarantine. One presented with clinical signs of anorexia and lethargy before death; the other died without prior clinical symptoms. The same Pasteurella-like organism was recovered antemortem from subcutaneous abscesses in two captive little golden mantled flying foxes (Pteropus pumilus) housed with additional E. wahlbergi. The organism was also cultured on tracheal wash from one Malaysian flying fox (Pteropus vampyrus) and another E. wahlbergi, both demonstrating clinical signs of pneumonia. All recovered isolates appeared morphologically and biochemically similar to the initial isolates and were further characterized as either a Pasteurella or Actinobacillus organism on the basis of biochemical and cellular fatty acid profiles. Screening of the current collection using pharyngeal swabs isolated this organism from 12 of 15 E. wahlbergi, two of three P. vampyrus, one of 26 island flying foxes (Pteropus hypomelanus), and one of nine Rodrigues fruit bats (Pteropus rodricensis). The organism was not identified in pharyngeal culture from eight Indian flying foxes (Pteropus giganteus), nine Egyptian fruit bats (Rousettus aegypticus), or an additional 16 P. pumilus.     

AVA letter sent to the Weekend Australian

The President of the AVA, Dr Garth McGilvray, last month sent the following letter to the Editor of the Weekend Australian newspaper. This was in response to inaccurate claims it had published earlier concerning the use of antibiotics in food production animals - and the risk of this leading to antibiotic resistance in humans. The letter was published unedited, except for the deletion of the last paragraph - apparently for space reasons.     

No Evidence of Hendra Virus Infection in the Australian Flying‐fox Ectoparasite Genus Cyclopodia

Hendra virus (HeV) causes potentially fatal respiratory and/or neurological disease in both horses and humans. Although Australian flying‐foxes of the genus Pteropus have been identified as reservoir hosts, the precise mechanism of HeV transmission has yet to be elucidated. To date, there has been limited investigation into the role of haematophagous insects as vectors of HeV. This mode of transmission is particularly relevant because Australian flying‐foxes host the bat‐specific blood‐feeding ectoparasites of the genus Cyclopodia (Diptera: Nycteribiidae), also known as bat flies. Using molecular detection methods, we screened for HeV RNA in 183 bat flies collected from flying‐foxes inhabiting a roost in Boonah, Queensland, Australia. It was subsequently demonstrated that during the study period, Pteropus alecto in this roost had a HeV RNA prevalence between 2 and 15% (95% CI [1, 6] to [8, 26], respectively). We found no evidence of HeV in any bat flies tested, including 10 bat flies collected from P. alecto in which we detected HeV RNA. Our negative findings are consistent with previous findings and provide additional evidence that bat flies do not play a primary role in HeV transmission.     

Infection with Menangle virus in flying foxes (Pteropus spp.) in Australia

Objective To examine flying foxes (Pteropus spp.) for evidence of infection with Menangle virus. Design Clustered non‐random sampling for serology, virus isolation and electron microscopy (EM). Procedure Serum samples were collected from 306 Pteropus spp. in northern and eastern Australia and tested for antibodies against Menangle virus (MenV) using a virus neutralisation test (VNT). Virus isolation was attempted from tissues and faeces collected from 215 Pteropus spp. in New South Wales. Faecal samples from 68 individual Pteropus spp. and four pools of faeces were examined by transmission EM following routine negative staining and immunogold labelling. Results Neutralising antibodies (VNT titres ≥ 8) against MenV were detected in 46% of black flying foxes (P. alecto), 41% of grey‐headed flying foxes (P. poliocephalus), 25% of spectacled flying foxes (P. conspicillatus) and 1% of little red flying foxes (P. scapulatus) in Australia. Positive sera included samples collected from P. poliocephalus in a colony adjacent to a piggery that had experienced reproductive disease caused by MenV. Virus‐like particles were observed by EM in faeces from Pteropus spp. and reactivity was detected in pooled faeces and urine by immunogold EM using sera from sows that had been exposed to MenV. Attempts to isolate the virus from the faeces and tissues from Pteropus spp. were unsuccessful. Conclusion Serological evidence of infection with MenV was detected in Pteropus spp. in Australia. Although virus‐like particles were detected in faeces, no viruses were isolated from faeces, urine or tissues of Pteropus spp.     

Faeces as a novel material to estimate lyssavirus prevalence in bat populations

Rabies is caused by infection with a lyssavirus. Bat rabies is of concern for both public health and bat conservation. The current method for lyssavirus prevalence studies in bat populations is by oral swabbing, which is invasive for the bats, dangerous for handlers, time‐consuming and expensive. In many situations, such sampling is not feasible, and hence, our understanding of epidemiology of bat rabies is limited. Faeces are usually easy to collect from bat colonies without disturbing the bats and thus could be a practical and feasible material for lyssavirus prevalence studies. To further explore this idea, we performed virological analysis on faecal pellets and oral swabs of seven serotine bats (Eptesicus serotinus) that were positive for European bat 1 lyssavirus in the brain. We also performed immunohistochemical and virological analyses on digestive tract samples of these bats to determine potential sources of lyssavirus in the faeces. We found that lyssavirus detection by RT‐qPCR was nearly as sensitive in faecal pellets (6/7 bats positive, 86%) as in oral swabs (7/7 bats positive, 100%). The likely source of lyssavirus in the faeces was virus excreted into the oral cavity from the salivary glands (5/6 bats positive by immunohistochemistry and RT‐qPCR) or tongue (3/4 bats positive by immunohistochemistry) and swallowed with saliva. Virus could not be isolated from any of the seven faecal pellets, suggesting the lyssavirus detected in faeces is not infectious. Lyssavirus detection in the majority of faecal pellets of infected bats shows that this novel material should be further explored for lyssavirus prevalence studies in bats.     

Plasma biochemistry and hematologic values for wild-caught flying foxes (Pteropus giganteus) in India.

Although bats of the genus Pteropus are important ecologically as pollinators and natural hosts for zoonotic pathogens, little is known about their basic physiology. Hematology and plasma biochemistries were determined from wild-caught flying foxes (Pteropus giganteus) in northern India (n=41). Mean lymphocyte differential count was higher for juveniles than adults. Mean platelet count was lower than previously reported. No hemoparasites were observed. No differences were observed between plasma biochemistry values of male and female bats, juveniles and adults, or lactating and nonlactating females. Variation in aspartate aminotransferase (AST) was seen based on body condition score. Blood urea nitrogen and cholesterol concentrations were lower in P. giganteus than other mammalian groups, but were consistent with those reported from other Pteropus species. Alanine aminotransferase and AST concentrations were higher than those reported for Pteropus vampyrus, a closely related species. This study provides basic physiologic information that can be used in future health and disease studies of Indian flying foxes.     

Hendra Virus Infection in Horses: A Review on Emerging Mystery Paramyxovirus

Hendra virus (HeV) is a zoonotic paramyxovirus which causes acute and deadly infection in horses (Equus caballus). It is a rare and unmanaged emerging viral infection in horses which is harbored by bats of the genus Pteropus (Australian flying foxes or fruit bats). The virus is pleomorphic in shape and its genome contains nonsegmented negative-stranded RNA with 18234 nucleotides in length. The virus is transmitted from flying foxes to horses, horse to horse, and horse to humans. Human-to-human transmission of HeV infection is not reported yet. The infection of HeV in horses is highly variable and shows broad range of signs and lesions including distinct respiratory and neurological disorders. Currently, there are no specific antiviral drugs available for the treatment of HeV infection in horses. Vaccination is considered as prime option to prevent HeV infection in horses. A subunit vaccine, called as “Equivac HeV vaccine” has been approved recently for preventing this viral infection in horses. In addition, a plethora of common preventive strategies could help restrict the inter- and intra-species transmission of HeV. Considering the scanty but severe fatality cases of this mystery virus as well as lack of proper attention by veterinary scientists, this review article spotlights not only on the clinical signs, transmission, epidemiology, biology, pathogenesis, and diagnosis of HeV but also the preventive managements of this uncommon infection in horses by vaccination and other precautious strategies.     

Detection of specific antibody responses to vaccination in variable flying foxes (Pteropus hypomelanus)

Megachiropteran bats are biologically important both as endangered species and reservoirs for emerging human pathogens. Reliable detection of antibodies to specific pathogens in bats is thus epidemiologically critical. Eight variable flying foxes (Pteropus hypomelanus) were immunized with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA). Each bat received monthly inoculations for 2 months. Affinity-purified IgG was used for production of polyclonal and monoclonal anti-variable flying fox IgG antibodies. ELISA and western blot analysis were used to monitor immune responses and for assessment of polyclonal and monoclonal antibody species cross-reactivity. Protein G, polyclonal antibodies, and monoclonal antibodies detected specific anti-DNP antibody responses in immunized variable flying foxes, with protein G being the most sensitive, followed by monoclonal antibodies and then polyclonal antibodies. While the polyclonal antibody was found to cross-react well against IgG of all bat species tested, some non-specific background was observed. The monoclonal antibody was found to cross-react well against IgG of six other species in the genus Pteropus and to cross-react less strongly against IgG from Eidolon helvum or Phyllostomus hastatus. Protein G distinguished best between vaccinated and unvaccinated bats, and these results validate the use of protein G for detection of bat IgG. Monoclonal antibodies developed in this study recognized immunoglobulins from other members of the genus Pteropus well, and may be useful in applications where specific detection of Pteropus IgG is needed.     

On pseudorabies in carnivores in Denmark. I. The red fox (Vulpes vulpes)

From January 1968 through February 1970 Pseudorabies was demonstrated in 12 red foxes (Vulpes vulpes) from different parts of Denmark. Eleven of them were free-living, while one had been kept in captivity.In nine foxes signs of intense local pruritus were found. Two of the remaining three foxes, which did not show such signs, had been killed in an acute phase of disease, while the last one had been found dead.The finding of remains of pigs in the digestive tract in some of the foxes, and the demonstration of Pseudorabies in piglets on a nearby farm in several cases, indicated that the foxes had caught the infection by eating dead piglets. The fox that had been kept in captivity had been fed with dead piglets which had shown signs of pseudorabies.The medulla oblongata and pons of these foxes were found to be the most suitable material for demonstration of psudorabies virus.     

The fox (Vulpes vulpes) as a reservoir for canine angiostrongylosis in Denmark. Field survey and experimental infections.

Until recently, Angiostrongylus vasorum was not considered to be an endogenous Danish parasite, since demonstration of this worm had been confined to necropsy findings in 2 dogs, both of which had visited France. During the last 2 years, however, clinical cases have been diagnosed among a considerable number of Danish dogs, none of which had ever been outside Denmark. All these cases have occurred north of Copenhagen, where an endemic focus seems to exist. In this field survey A. vasorum was found for the first time in wild Danish red foxes. Furthermore, experimental infections showed that the parasite can be transferred between foxes and dogs. Consequently, the wild fox population must be considered a potential reservoir for transfer of A. vasorum to domestic dogs.     

Echinococcus multilocularis (Cestoda,Taeniidae) in Red foxes (Vulpes vulpes) in northern Belgium

The first record of the tapeworm Echinococcus multilocularis (Cestoda, Taeniidae) in Red foxes (Vulpes vulpes) in northern Belgium is described. Between 1996 and 1999, 237 dead foxes were examined for the presence of this tapeworm using the intestinal scraping technique. Four foxes (1.7%) were found to be infected with E. multilocularis and showed medium to very high parasitic burdens. Three infected foxes originated from the south of the study area and the fourth animal came from the north of the study area near the border with The Netherlands. These findings are discussed in relation to the high endemicity of E. multilocularis in southern Belgium and to the increased distribution of the Red fox (V. vulpes) in northern Belgium during the last two decades.     

Screening for infection of Trichinella in red fox (Vulpes vulpes) in Denmark

A total of 6141 foxes (Vulpes vulpes) were examined for infection with Trichinella. The foxes were killed in Denmark during the hunting season 1995-1996 and 1997-1998; 3133 and 3008, respectively. Foxes included in the investigation came from throughout the country with the exception of the island of Bornholm. The right foreleg from each fox was submitted for investigation. The legs were stored at -20 degrees C for 3-10 months prior to examination. Following thawing, muscle tissue (10 g) from each leg was examined by trichinoscopy and by a pepsin-HCl digestion technique. In 1995-1996, three foxes were found positive corresponding to a prevalence of 0.001. Each of the infected foxes harboured an extremely low infection, i.e. about one larva per 10 g muscle tissue. It was not possible to obtain sufficient larval material for species identification. All three foxes were shot in the vicinity of a small village in the north-western part of Denmark. In 1997-1998 no Trichinella cases were found. The results, compared with previous studies, indicate that the prevalence of infection of Trichinella sp. among wild living foxes in Denmark is very low. This is further supported by the fact, that no infection of Trichinella sp. has been found in slaughtered pigs in Denmark for more than 65 years, which suggests that the infection pressure is very low. Considering the facts above we conclude that the risk of Trichinella infections is negligible in intensive indoor pig production units in Denmark whereas high local prevalence of Trichinella infections in the wildlife might constitute a serious risk for the expanding outdoor pig production.